angiotensin ii angii  (Millipore)


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    Name:
    Anti GABA antibody
    Description:
    Anti GABA is produced in rabbit using GABA BSA as the immunogen The antibody is isolated from antiserum by immunospecific methods of purification Antigen specific affinity isolation removes essentially all rabbit serum proteins including immunoglobulins which do not specifically bind to GABA GABA γ aminobutyric acid is a multifunctional molecule found in several organisms from prokaryotes to vertebrates It is present in non nervous structures such as peripheral nervous and endocrine systems GABA is formed following decarboxylation of glutamic acid by the enzyme glutamic acid decarboxylase GAD
    Catalog Number:
    a2052
    Price:
    None
    Applications:
    Rabbit anti-GABA antibody has been used for electron microscopy analysis in rat hippocampal tissues at a dilution of 1:4000. The antibody has also been used for immunocytochemistry applications at dilutions ranging from 1:100-1:750 in Drosophila and Periplaneta americana brain cells.
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    Structured Review

    Millipore angiotensin ii angii
    Anti GABA antibody
    Anti GABA is produced in rabbit using GABA BSA as the immunogen The antibody is isolated from antiserum by immunospecific methods of purification Antigen specific affinity isolation removes essentially all rabbit serum proteins including immunoglobulins which do not specifically bind to GABA GABA γ aminobutyric acid is a multifunctional molecule found in several organisms from prokaryotes to vertebrates It is present in non nervous structures such as peripheral nervous and endocrine systems GABA is formed following decarboxylation of glutamic acid by the enzyme glutamic acid decarboxylase GAD
    https://www.bioz.com/result/angiotensin ii angii/product/Millipore
    Average 89 stars, based on 1819 article reviews
    Price from $9.99 to $1999.99
    angiotensin ii angii - by Bioz Stars, 2020-09
    89/100 stars

    Images

    1) Product Images from "ClC-2 knockdown prevents cerebrovascular remodeling via inhibition of the Wnt/β-catenin signaling pathway"

    Article Title: ClC-2 knockdown prevents cerebrovascular remodeling via inhibition of the Wnt/β-catenin signaling pathway

    Journal: Cellular & Molecular Biology Letters

    doi: 10.1186/s11658-018-0095-z

    Lack of ClC-2 reduced AngII-induced HBVSMC proliferation. a and b Cells were transfected with ClC-2 siRNA (siClC-2; 20 nM) or negative siRNA (negative; 20 nM) for 48 h in prior to angiotensin II (AngII) treatment (10 − 7 M) for another 48 h. Cell proliferation was determined using the CCK-8 assay ( a ) and BrdU incorporation ( b ). c and d The protein expressions of PCNA ( c ) and Ki67 ( d ) were detected using western blotting. ** p
    Figure Legend Snippet: Lack of ClC-2 reduced AngII-induced HBVSMC proliferation. a and b Cells were transfected with ClC-2 siRNA (siClC-2; 20 nM) or negative siRNA (negative; 20 nM) for 48 h in prior to angiotensin II (AngII) treatment (10 − 7 M) for another 48 h. Cell proliferation was determined using the CCK-8 assay ( a ) and BrdU incorporation ( b ). c and d The protein expressions of PCNA ( c ) and Ki67 ( d ) were detected using western blotting. ** p

    Techniques Used: Transfection, CCK-8 Assay, BrdU Incorporation Assay, Western Blot

    2) Product Images from "Wavy Multistratified Amacrine Cells in the Monkey Retina Contain Immunoreactive Secretoneurin"

    Article Title: Wavy Multistratified Amacrine Cells in the Monkey Retina Contain Immunoreactive Secretoneurin

    Journal: Peptides

    doi: 10.1016/j.peptides.2017.06.005

    Interactions between dendrites of secretoneurin-IR amacrine cells (red) and melanopsin-IR (green) retinal ganglion cells. Secretoneurin-IR dendrites are closely apposed to dendrites of outer-stratifying melanopsin cells (arrowheads). A. Note that a secretoneurin-IR dendrite also contacts the soma of an outer melanopsin cell. The main figure is an orthogonal projection of 6 optical sections showing only the melanopsin signal (green), z step = 0.5 μm, scale bar = 20 μm. Insets are single optical sections displaying both melanopsin (green) and secretoneurin (red) signals, scale bars = 2 μm. B. Note the co-fasciculation of the 2 dendrites. The top figure is an orthogonal projection of 10 optical sections, z step = 0.31 μm. The others are consecutive single optical sections. Scale bar = 5 μm.
    Figure Legend Snippet: Interactions between dendrites of secretoneurin-IR amacrine cells (red) and melanopsin-IR (green) retinal ganglion cells. Secretoneurin-IR dendrites are closely apposed to dendrites of outer-stratifying melanopsin cells (arrowheads). A. Note that a secretoneurin-IR dendrite also contacts the soma of an outer melanopsin cell. The main figure is an orthogonal projection of 6 optical sections showing only the melanopsin signal (green), z step = 0.5 μm, scale bar = 20 μm. Insets are single optical sections displaying both melanopsin (green) and secretoneurin (red) signals, scale bars = 2 μm. B. Note the co-fasciculation of the 2 dendrites. The top figure is an orthogonal projection of 10 optical sections, z step = 0.31 μm. The others are consecutive single optical sections. Scale bar = 5 μm.

    Techniques Used:

    3) Product Images from "Human chondrogenic paraxial mesoderm, directed specification and prospective isolation from pluripotent stem cells"

    Article Title: Human chondrogenic paraxial mesoderm, directed specification and prospective isolation from pluripotent stem cells

    Journal: Scientific Reports

    doi: 10.1038/srep00455

    Prospective isolation of paraxial mesoderm and demonstration of their chondrogenic activity. (A, B) Enrichment of paraxial mesoderm generated from H9 hES cells. H9 hES cells were differentiated under BION conditions, and total progeny (Pre) and the KDR − PDGFRα + (P+, red gate in A) and KDR − PDGFRα − (P-, blue gate in A) progeny were isolated by FACS and subjected to RT-PCR (B). (C) MEOX1 protein expression. Mesodermal progeny, derived from H9 hES cells in the presence of BIO + Noggin (BION) or BIO alone (BIO) and cultured on a chamber slide for 5 day, were immunostained with the anti-MEOX1 antibody. The nucleoli staining pink within a blue nucleus, some of which are indicated with white arrows, are detectable in progeny generated under the BION condition. Isotype controls are shown in Supplementary Fig. 1C. Scale Bars, 50 μm. (D) Time-course of FACS analysis for MIXL1-GFP (GFP), KDR and PDGFRα expression during Mixl1-GFP hES cell differentiation induced in the presence of BIO + SB + Noggin (BIOSN). (E, F) Enrichment of paraxial mesoderm derived from other hPS cells. Mixl1-GFP hES cells and HK1 hiPS cells were differentiated under BIOSN conditions. From Mixl1-GFP hES cells, total EB cells (Pre) and the GFP + KDR − PDGFRα + (GFP+P+ red gate) and GFP + KDR − PDGFRα − (GFP+P− blue gate) progeny were isolated by FACS (E) and subjected to RT-PCR using the MEOX1 primers (F). From HK1 hiPS cells, only the KDR − PDGFRα + (P+) and KDR − PDGFRα − (P−) progeny were accumulated (E) as in the case of H9 hES cells.
    Figure Legend Snippet: Prospective isolation of paraxial mesoderm and demonstration of their chondrogenic activity. (A, B) Enrichment of paraxial mesoderm generated from H9 hES cells. H9 hES cells were differentiated under BION conditions, and total progeny (Pre) and the KDR − PDGFRα + (P+, red gate in A) and KDR − PDGFRα − (P-, blue gate in A) progeny were isolated by FACS and subjected to RT-PCR (B). (C) MEOX1 protein expression. Mesodermal progeny, derived from H9 hES cells in the presence of BIO + Noggin (BION) or BIO alone (BIO) and cultured on a chamber slide for 5 day, were immunostained with the anti-MEOX1 antibody. The nucleoli staining pink within a blue nucleus, some of which are indicated with white arrows, are detectable in progeny generated under the BION condition. Isotype controls are shown in Supplementary Fig. 1C. Scale Bars, 50 μm. (D) Time-course of FACS analysis for MIXL1-GFP (GFP), KDR and PDGFRα expression during Mixl1-GFP hES cell differentiation induced in the presence of BIO + SB + Noggin (BIOSN). (E, F) Enrichment of paraxial mesoderm derived from other hPS cells. Mixl1-GFP hES cells and HK1 hiPS cells were differentiated under BIOSN conditions. From Mixl1-GFP hES cells, total EB cells (Pre) and the GFP + KDR − PDGFRα + (GFP+P+ red gate) and GFP + KDR − PDGFRα − (GFP+P− blue gate) progeny were isolated by FACS (E) and subjected to RT-PCR using the MEOX1 primers (F). From HK1 hiPS cells, only the KDR − PDGFRα + (P+) and KDR − PDGFRα − (P−) progeny were accumulated (E) as in the case of H9 hES cells.

    Techniques Used: Isolation, Activity Assay, Generated, FACS, Reverse Transcription Polymerase Chain Reaction, Expressing, Derivative Assay, Cell Culture, Staining, Cell Differentiation

    Quantitative monitoring of chondrogenesis from hPS cell-derived paraxial mesoderm and adult hMSCs. ( A ) 3D pellet chondrogenesis culture conditions for the sorted hPS cell progeny and the STRO1 + hMSCs. (B) Cartilage particle formation from the KDR − PDGFRα + paraxial mesoderm-derived from H9 and Mix1-GFP hES cells and HK1 hiPS cells. Chondrogenesis from the KDR − PDGFRα + progeny (P+ cells, Fig. 3A ) from H9 hES cells was performed under P ( a ), PT ( b ), PB ( c ), and PTB ( d–h ) conditions in 3D pellet culture for 24 days. The GFP + KDR − PDGFRα + progeny ( e , f ) and GFP + KDR − PDGFRα − progeny ( g ) from Mixl1-GFP hES cells (GFP+P+ and GFP+P− cells, respectively, Fig. 3E ) and the KDR − PDGFRα + progeny (P+ cells, Fig. 3E ) from HK1 hiPS cells ( h ) were also pellet-cultured under PTB conditions. The resulting cartilage particles were paraffin-sectioned, and stained with Toluidine blue ( a–e , g , h ) or immunostained with the anti-COL2 antibody ( f ). The arrow indicates a small cartilage nodule. Scale bars, 500 μm. (C) The COL2/COL1 protein ratio. The conditioned media of the cartilage particles formed under PT, PTB, PT/B, T, or TB conditions as indicated, using the KDR − PDGFRα + paraxial mesoderm from H9 hES cells and HK1 hiPS cells, and the STRO1 + hMSCs from the bone marrow of donor 1 (STRO1 + MSC1), were subjected to COL1 and COL2 ELISA. The ratio ng COL2/ng COL1 was calculated, averaged and plotted (shown as a bar). The SD is shown as a thin vertical line. (D) Change in the level of COL10 transcript in the H9 hES cell-derived KDR − PDGFRα + paraxial mesoderm before (Sorted P + ) and after 3D pellet culture performed under PT and PTB conditions.
    Figure Legend Snippet: Quantitative monitoring of chondrogenesis from hPS cell-derived paraxial mesoderm and adult hMSCs. ( A ) 3D pellet chondrogenesis culture conditions for the sorted hPS cell progeny and the STRO1 + hMSCs. (B) Cartilage particle formation from the KDR − PDGFRα + paraxial mesoderm-derived from H9 and Mix1-GFP hES cells and HK1 hiPS cells. Chondrogenesis from the KDR − PDGFRα + progeny (P+ cells, Fig. 3A ) from H9 hES cells was performed under P ( a ), PT ( b ), PB ( c ), and PTB ( d–h ) conditions in 3D pellet culture for 24 days. The GFP + KDR − PDGFRα + progeny ( e , f ) and GFP + KDR − PDGFRα − progeny ( g ) from Mixl1-GFP hES cells (GFP+P+ and GFP+P− cells, respectively, Fig. 3E ) and the KDR − PDGFRα + progeny (P+ cells, Fig. 3E ) from HK1 hiPS cells ( h ) were also pellet-cultured under PTB conditions. The resulting cartilage particles were paraffin-sectioned, and stained with Toluidine blue ( a–e , g , h ) or immunostained with the anti-COL2 antibody ( f ). The arrow indicates a small cartilage nodule. Scale bars, 500 μm. (C) The COL2/COL1 protein ratio. The conditioned media of the cartilage particles formed under PT, PTB, PT/B, T, or TB conditions as indicated, using the KDR − PDGFRα + paraxial mesoderm from H9 hES cells and HK1 hiPS cells, and the STRO1 + hMSCs from the bone marrow of donor 1 (STRO1 + MSC1), were subjected to COL1 and COL2 ELISA. The ratio ng COL2/ng COL1 was calculated, averaged and plotted (shown as a bar). The SD is shown as a thin vertical line. (D) Change in the level of COL10 transcript in the H9 hES cell-derived KDR − PDGFRα + paraxial mesoderm before (Sorted P + ) and after 3D pellet culture performed under PT and PTB conditions.

    Techniques Used: Derivative Assay, Cell Culture, Staining, Enzyme-linked Immunosorbent Assay

    Factor-dependent chondrogenesis from the KDR − PDGFRα + paraxial mesoderm-like cells from various hPS cell lines. (A) Chondrogenesis from the H9 hES cell-derived KDR − PDGFRα + paraxial mesoderm performed under P ( a ), PT ( c ), PB ( d ), and PTB ( d ) conditions in 2D micromass culture for 16 days. (Left) Period of treatment with PDGF (P, red), TGFβ3 (T, blue) and/or BMP4 (B, green) is indicated as a bar. (Right) The resulting micromasses were stained with acid Alcian Blue. Arrows indicate small cartilage nodules. Scale bars, 500 μm. (B) Expression of chondrogenesis-specific genes during 2D micromass culture of the H9 hES cell-derived KDR − PDGFRα − paraxial mesodermal cells performed under P, PT, PB or PTB conditions. The resulting micromasses were subjected to RT-PCR on the day indicated with primers for the genes indicated.
    Figure Legend Snippet: Factor-dependent chondrogenesis from the KDR − PDGFRα + paraxial mesoderm-like cells from various hPS cell lines. (A) Chondrogenesis from the H9 hES cell-derived KDR − PDGFRα + paraxial mesoderm performed under P ( a ), PT ( c ), PB ( d ), and PTB ( d ) conditions in 2D micromass culture for 16 days. (Left) Period of treatment with PDGF (P, red), TGFβ3 (T, blue) and/or BMP4 (B, green) is indicated as a bar. (Right) The resulting micromasses were stained with acid Alcian Blue. Arrows indicate small cartilage nodules. Scale bars, 500 μm. (B) Expression of chondrogenesis-specific genes during 2D micromass culture of the H9 hES cell-derived KDR − PDGFRα − paraxial mesodermal cells performed under P, PT, PB or PTB conditions. The resulting micromasses were subjected to RT-PCR on the day indicated with primers for the genes indicated.

    Techniques Used: Derivative Assay, Staining, Expressing, Reverse Transcription Polymerase Chain Reaction

    Analysis of comparative cartilage-formation by hES cell-derived paraxial mesoderm-like cells and adult bone marrow-derived hMSCs. The H9 hES cell-derived KDR − PDGFRα + paraxial mesoderm (A–C) and human bone marrow-derived STRO1 + MSCs from donor 1 (p4, STRO1 + MSC1) (D–G) were subjected to 3D pellet chondrogenesis culture under PT (A, F), PTB (B, G), PT/B (C), T (D), and TB (E) conditions. Four contiguous sections were stained with Toluidine Blue (T-Blue) (i, ii), or immunostained for COL1 (iii), COL2 (iv), or isotype control (v). (ii) shows an enlarged view of the square in (i). Scale bars, 200 μm.
    Figure Legend Snippet: Analysis of comparative cartilage-formation by hES cell-derived paraxial mesoderm-like cells and adult bone marrow-derived hMSCs. The H9 hES cell-derived KDR − PDGFRα + paraxial mesoderm (A–C) and human bone marrow-derived STRO1 + MSCs from donor 1 (p4, STRO1 + MSC1) (D–G) were subjected to 3D pellet chondrogenesis culture under PT (A, F), PTB (B, G), PT/B (C), T (D), and TB (E) conditions. Four contiguous sections were stained with Toluidine Blue (T-Blue) (i, ii), or immunostained for COL1 (iii), COL2 (iv), or isotype control (v). (ii) shows an enlarged view of the square in (i). Scale bars, 200 μm.

    Techniques Used: Derivative Assay, Staining

    Directed specification of paraxial mesoderm in a chemically defined medium. (A) Differentiation protocol for hPS cells. (B, C) GSK3 inhibitor and Noggin-dependent specification of paraxial mesoderm and associated changes in the progenitor profile after 8 days of differentiation of H9 hES cells. (B) FACS analysis. (C) RT-PCR with MEOX1 and TCF15 primers. No, None; N, Noggin; AceBIO/Ace-BIO, Acetoxime-BIO; CHIR, CHIR99021; MeBIO, Methyl-BIO. (D) Time-dependent differentiation of H9 hES cells in the presence of no factors (No), BIO, and BIO + Noggin (BION). Specification of mesoderm was monitored by RT-PCR.
    Figure Legend Snippet: Directed specification of paraxial mesoderm in a chemically defined medium. (A) Differentiation protocol for hPS cells. (B, C) GSK3 inhibitor and Noggin-dependent specification of paraxial mesoderm and associated changes in the progenitor profile after 8 days of differentiation of H9 hES cells. (B) FACS analysis. (C) RT-PCR with MEOX1 and TCF15 primers. No, None; N, Noggin; AceBIO/Ace-BIO, Acetoxime-BIO; CHIR, CHIR99021; MeBIO, Methyl-BIO. (D) Time-dependent differentiation of H9 hES cells in the presence of no factors (No), BIO, and BIO + Noggin (BION). Specification of mesoderm was monitored by RT-PCR.

    Techniques Used: FACS, Reverse Transcription Polymerase Chain Reaction

    4) Product Images from "The Somatostatin 2A Receptor Is Enriched in Migrating Neurons during Rat and Human Brain Development and Stimulates Migration and Axonal Outgrowth"

    Article Title: The Somatostatin 2A Receptor Is Enriched in Migrating Neurons during Rat and Human Brain Development and Stimulates Migration and Axonal Outgrowth

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0005509

    Regional and cellular distribution of sst2A receptor immunoreactivity in the human developing cortex. A,B) Embryonic sagittal sections at GW 8 at the level of the medial cerebral cortex (A) reveals numerous receptor immunoreactive cell bodies (purple color) in the preplate (PP) (inset box) and in the subventricular zone (SVZ). By contrast the ventricular zone (VZ) is devoid of receptor immunoreactivity. The red color is due to the counterstaining of sections with neutral red. In the lateral part of the medial cerebral cortex (B), sst2A receptor immunoreactivity is detected in the marginal zone (MZ), cortical plate (CP), subplate/intermediate zone (SP/IZ) and SVZ (inset box). C–E) At GW 12 in coronal sections, albeit less intense, the pattern of receptor immunoreactivity is comparable to that observed at GW 8 with higher signals in the MZ (C,D) and SVZ (C,E). D and E are magnifications of boxed areas in C at the level of the MZ and the SVZ, respectively. Note in D that some bipolar neurons expressing the sst2A receptor are visible in the CP and in E that patches of labeling are observed in the SVZ contiguous to the VZ. F, G) In coronal sections at GW 23, the labeling is present in neurons of CP (F) as well as in presumably post-mitotic migrating neurons in the IZ (G). H–J) In coronal sections at birth, the labeling is diffusely distributed in layers II–III and V. In this latter layer some neurons positive for the sst2A receptor are also observed (I,J). Scale bars: A, B, D, E, G, 25 µm; C, F, H, 100 µm; Inset in A,B and I,J, 10 µm.
    Figure Legend Snippet: Regional and cellular distribution of sst2A receptor immunoreactivity in the human developing cortex. A,B) Embryonic sagittal sections at GW 8 at the level of the medial cerebral cortex (A) reveals numerous receptor immunoreactive cell bodies (purple color) in the preplate (PP) (inset box) and in the subventricular zone (SVZ). By contrast the ventricular zone (VZ) is devoid of receptor immunoreactivity. The red color is due to the counterstaining of sections with neutral red. In the lateral part of the medial cerebral cortex (B), sst2A receptor immunoreactivity is detected in the marginal zone (MZ), cortical plate (CP), subplate/intermediate zone (SP/IZ) and SVZ (inset box). C–E) At GW 12 in coronal sections, albeit less intense, the pattern of receptor immunoreactivity is comparable to that observed at GW 8 with higher signals in the MZ (C,D) and SVZ (C,E). D and E are magnifications of boxed areas in C at the level of the MZ and the SVZ, respectively. Note in D that some bipolar neurons expressing the sst2A receptor are visible in the CP and in E that patches of labeling are observed in the SVZ contiguous to the VZ. F, G) In coronal sections at GW 23, the labeling is present in neurons of CP (F) as well as in presumably post-mitotic migrating neurons in the IZ (G). H–J) In coronal sections at birth, the labeling is diffusely distributed in layers II–III and V. In this latter layer some neurons positive for the sst2A receptor are also observed (I,J). Scale bars: A, B, D, E, G, 25 µm; C, F, H, 100 µm; Inset in A,B and I,J, 10 µm.

    Techniques Used: Expressing, Labeling

    Regional and cellular localization of the sst2A receptor immunoreactivity in sagittal sections of the rat rhombencephalon at embryonic day 13 (E13). A) Densely packed sst2A receptor-immunoreactive cells are observed in the marginal zone contiguous to the ventricular zone of the rhombomeres (r1 to r6, arrowheads) and in the lateral reticular formation (LRtF). B–B″) Sst2A receptor-immunoreactive cells (red) are localized in the marginal zone (B) whereas proliferating cells identified by the proliferation marker Ki-67 (green) are concentrated in the ventricular zone (B′). The lack of overlap between the two signals (B″) indicates that sst2A receptor-expressing cells are predominantly post-mitotic. C) The majority of sst2A receptor-immunoreactive cells have small round perikarya and some exhibit immunolabeled processes that are oriented perpendicularly to the ventricular surface. D) A few sst2A receptor-immunoreactive cells are bipolar, displaying the morphological features of migrating neurons. E) In the LRtF, cell bodies are strongly sst2A receptor-immunoreactive. F–F″) An sst2A receptor-immunoreactive cell (red in F, F″) of the LRtF is found to be Ki-67-positive (green in F′, F″) (arrowheads). The low percentage of colocalization (F″) indicates that the majority of receptor-expressing cells are post-mitotic. G–G″) The post-mitotic feature of most sst2A receptor-immunoreactive cells (red in G, G″) of the rhombencephalon is further indicated by the colocalization (G″) with the post-mitotic neuronal marker β-tubulin (green in G′, G″) (arrowheads), as illustrated in the facial nucleus. Scale bars: A, 250 µm; B–B″, G–G″, 50 µm; C, E, 20 µm; F, 10 µm.
    Figure Legend Snippet: Regional and cellular localization of the sst2A receptor immunoreactivity in sagittal sections of the rat rhombencephalon at embryonic day 13 (E13). A) Densely packed sst2A receptor-immunoreactive cells are observed in the marginal zone contiguous to the ventricular zone of the rhombomeres (r1 to r6, arrowheads) and in the lateral reticular formation (LRtF). B–B″) Sst2A receptor-immunoreactive cells (red) are localized in the marginal zone (B) whereas proliferating cells identified by the proliferation marker Ki-67 (green) are concentrated in the ventricular zone (B′). The lack of overlap between the two signals (B″) indicates that sst2A receptor-expressing cells are predominantly post-mitotic. C) The majority of sst2A receptor-immunoreactive cells have small round perikarya and some exhibit immunolabeled processes that are oriented perpendicularly to the ventricular surface. D) A few sst2A receptor-immunoreactive cells are bipolar, displaying the morphological features of migrating neurons. E) In the LRtF, cell bodies are strongly sst2A receptor-immunoreactive. F–F″) An sst2A receptor-immunoreactive cell (red in F, F″) of the LRtF is found to be Ki-67-positive (green in F′, F″) (arrowheads). The low percentage of colocalization (F″) indicates that the majority of receptor-expressing cells are post-mitotic. G–G″) The post-mitotic feature of most sst2A receptor-immunoreactive cells (red in G, G″) of the rhombencephalon is further indicated by the colocalization (G″) with the post-mitotic neuronal marker β-tubulin (green in G′, G″) (arrowheads), as illustrated in the facial nucleus. Scale bars: A, 250 µm; B–B″, G–G″, 50 µm; C, E, 20 µm; F, 10 µm.

    Techniques Used: Marker, Expressing, Immunolabeling

    Expression of the sst2A receptor in serotoninergic neurons of the rat brainstem at E16. A) Triple-labeling with sst2A receptor (red), 5-HT (green) and DAPI (blue) in the ventral part of the brainstem illustrates that most serotoninergic neurons are sst2A receptor immunoreactive. B,B′ represent magnification of the boxed area in A. Note the extensive colocalization of sst2A receptor and 5-HT in both cell bodies and processes. C) The sst2A receptor is also expressed in serotoninergic migrating cells in the more dorsal part of the mesencephalon presumably corresponding to the dorsal raphe nucleus. D,D′ represents magnification of the boxed area in C and illustrates double-labeling in both the soma and processes of migrating neurons. Scale bars: A, C, 50 µm; B, D, 20 µm.
    Figure Legend Snippet: Expression of the sst2A receptor in serotoninergic neurons of the rat brainstem at E16. A) Triple-labeling with sst2A receptor (red), 5-HT (green) and DAPI (blue) in the ventral part of the brainstem illustrates that most serotoninergic neurons are sst2A receptor immunoreactive. B,B′ represent magnification of the boxed area in A. Note the extensive colocalization of sst2A receptor and 5-HT in both cell bodies and processes. C) The sst2A receptor is also expressed in serotoninergic migrating cells in the more dorsal part of the mesencephalon presumably corresponding to the dorsal raphe nucleus. D,D′ represents magnification of the boxed area in C and illustrates double-labeling in both the soma and processes of migrating neurons. Scale bars: A, C, 50 µm; B, D, 20 µm.

    Techniques Used: Expressing, Labeling

    Distribution of the sst2A receptor immunoreactivity in the rat hippocampus during pre- and postnatal development. A–A″) At E16, sst2A receptor immunoreactivity (red in A, A″) is localized in the intermediate zone (IZ) of the hippocampus. Note the lack of immunoreactivity in the ventricular zone (VZ). B–B″) At E21, the most intense immunolabeling is found in the intermediate zone of CA1. In addition, less intense immunolabeling is apparent in the pyramidal cell layer as well as in the strata oriens and radiatum of CA1, in the CA3 and in the developing dentate gyrus (DG). C) In the hilus of the DG, sst2A receptor immunoreactivity appears diffusely distributed. D represents magnification of the area labeled with asterisk in B and illustrates the diffuse sst2A receptor immunolabeling observed in the CA1 pyramidal cell layer. E represents magnification of boxed area in B. The sst2A receptor immunolabeling is intense in cells of the IZ whereas the subventricular zone (SVZ) is devoid of labeling. F) At P3, intense immunofluorescence is detected in the pyramidal layer, strata oriens and radiatum of CA1-3, as well as in the hilus of DG. The molecular layer of dentate gyrus is weakly immunoreactive. G represents magnification of boxed area in F and illustrates the intense sst2A receptor immunolabeling localized in CA1 pyramidal cell bodies and proximal dendrites. H represents magnification of area labeled with asterisk in F and illustrates the diffuse sst2A receptor immunolabeling observed in the hilus of the DG. Scale bars: A–A″, B–B″, F, 100 µm; C, D, E, G, H, 20 µm.
    Figure Legend Snippet: Distribution of the sst2A receptor immunoreactivity in the rat hippocampus during pre- and postnatal development. A–A″) At E16, sst2A receptor immunoreactivity (red in A, A″) is localized in the intermediate zone (IZ) of the hippocampus. Note the lack of immunoreactivity in the ventricular zone (VZ). B–B″) At E21, the most intense immunolabeling is found in the intermediate zone of CA1. In addition, less intense immunolabeling is apparent in the pyramidal cell layer as well as in the strata oriens and radiatum of CA1, in the CA3 and in the developing dentate gyrus (DG). C) In the hilus of the DG, sst2A receptor immunoreactivity appears diffusely distributed. D represents magnification of the area labeled with asterisk in B and illustrates the diffuse sst2A receptor immunolabeling observed in the CA1 pyramidal cell layer. E represents magnification of boxed area in B. The sst2A receptor immunolabeling is intense in cells of the IZ whereas the subventricular zone (SVZ) is devoid of labeling. F) At P3, intense immunofluorescence is detected in the pyramidal layer, strata oriens and radiatum of CA1-3, as well as in the hilus of DG. The molecular layer of dentate gyrus is weakly immunoreactive. G represents magnification of boxed area in F and illustrates the intense sst2A receptor immunolabeling localized in CA1 pyramidal cell bodies and proximal dendrites. H represents magnification of area labeled with asterisk in F and illustrates the diffuse sst2A receptor immunolabeling observed in the hilus of the DG. Scale bars: A–A″, B–B″, F, 100 µm; C, D, E, G, H, 20 µm.

    Techniques Used: Immunolabeling, Labeling, Immunofluorescence

    Regional, cellular and subcellular distribution of sst2A receptor immunoreactivity on coronal sections of the rat telencephalon at E16 and E18. A,B) Intense sst2A receptor immunoreactivity is detected at E16 in the post-mitotic areas of the lateral ganglionic eminence (LGE) and the caudal ganglionic eminence (CGE) (B). Note the presence of sst2A receptor immunoreactivity in the cortex (cx) and hippocampus (hi). C represents magnification of boxed area in B and illustrates that the sst2A receptor immunoreactivity is found in cell bodies and short processes in the CGE. D,E) Pre-embedding immunogold immunohistochemistry of the sst2A receptor in the CGE illustrates that high density of immunoparticles are localized intracellularly. However, sst2A receptor-immunoreactive particles are also found in association with the plasma membrane (arrowheads in D). Note that in a neuronal process the majority of the immunoparticles are membrane-associated (arrowheads in E). F represents magnification of the boxed area in A. Numerous cells are immunoreactive for sst2A in the LGE. G represents high magnification of the area labeled with an arrow on A and illustrates that fibers are also sst2A receptor-immunolabeled. H,I) High magnification confocal microscopic analysis in the CGE demonstrates redistribution of receptors upon agonist stimulation. In control conditions, sst2A receptor immunoreactivity outlines the periphery of cells (H). Forty minutes after agonist administration, receptor immunoreactivity is confined to small puncta in the cytoplasm (I). J,K) At E18, intense sst2A receptor immunoreactivity is observed in the dorso-medial part of the caudate-putamen in rostral (J) and caudal (K) sections close to the ventricular surface. Scattered sst2A receptor immunoreactivity is also evident in the medial part of the developing caudate-putamen (asterisk). L represents magnification of boxed area on J. The sst2A receptor immunoreactivity is observed in large number of cells and their short processes in the dorsal caudate-putamen. Note the lack of sst2A receptor immunoreactivity in the subventricular zone (SVZ). M,N are high magnifications from the area labeled with asterisk on J. The sst2A receptor is expressed in neuronal perikarya and processes in the medial part of the caudate-putamen. Scale bars: A, B, 200 µm; C, F, G, L, 20 µm; D, 500 nm; E, 250 nm; H, I, M, N, 10 µm; J, K, 500 µm.
    Figure Legend Snippet: Regional, cellular and subcellular distribution of sst2A receptor immunoreactivity on coronal sections of the rat telencephalon at E16 and E18. A,B) Intense sst2A receptor immunoreactivity is detected at E16 in the post-mitotic areas of the lateral ganglionic eminence (LGE) and the caudal ganglionic eminence (CGE) (B). Note the presence of sst2A receptor immunoreactivity in the cortex (cx) and hippocampus (hi). C represents magnification of boxed area in B and illustrates that the sst2A receptor immunoreactivity is found in cell bodies and short processes in the CGE. D,E) Pre-embedding immunogold immunohistochemistry of the sst2A receptor in the CGE illustrates that high density of immunoparticles are localized intracellularly. However, sst2A receptor-immunoreactive particles are also found in association with the plasma membrane (arrowheads in D). Note that in a neuronal process the majority of the immunoparticles are membrane-associated (arrowheads in E). F represents magnification of the boxed area in A. Numerous cells are immunoreactive for sst2A in the LGE. G represents high magnification of the area labeled with an arrow on A and illustrates that fibers are also sst2A receptor-immunolabeled. H,I) High magnification confocal microscopic analysis in the CGE demonstrates redistribution of receptors upon agonist stimulation. In control conditions, sst2A receptor immunoreactivity outlines the periphery of cells (H). Forty minutes after agonist administration, receptor immunoreactivity is confined to small puncta in the cytoplasm (I). J,K) At E18, intense sst2A receptor immunoreactivity is observed in the dorso-medial part of the caudate-putamen in rostral (J) and caudal (K) sections close to the ventricular surface. Scattered sst2A receptor immunoreactivity is also evident in the medial part of the developing caudate-putamen (asterisk). L represents magnification of boxed area on J. The sst2A receptor immunoreactivity is observed in large number of cells and their short processes in the dorsal caudate-putamen. Note the lack of sst2A receptor immunoreactivity in the subventricular zone (SVZ). M,N are high magnifications from the area labeled with asterisk on J. The sst2A receptor is expressed in neuronal perikarya and processes in the medial part of the caudate-putamen. Scale bars: A, B, 200 µm; C, F, G, L, 20 µm; D, 500 nm; E, 250 nm; H, I, M, N, 10 µm; J, K, 500 µm.

    Techniques Used: Hi-C, Immunohistochemistry, Labeling, Immunolabeling

    Regional and cellular distribution of the sst2A receptor immunofluorescence in the human cerebral cortex at GW 19. A–A″) Intensely labeled sst2A receptor-immunoreactive neurons (red in A′–A″) form chain-like clusters in the middle part of the cortical plate (CP). Note that long sst2A receptor-immunoreactive radial processes reach the pial surface (PS). B–B″) Receptor-immunolabeled cells and processes (red in B, B″) are closely apposed by vimentin-positive processes (green in B′B″), suggesting migration of sst2A-labeled cells on radial glia. C–C″) Sst2A receptor-immunoreactive processes (red in C, C″) are contacted by fibers that are immunoreactive for SRIF (green in D, D″) (arrowheads), the endogen ligand of the receptor. Scale bars: 20 µm.
    Figure Legend Snippet: Regional and cellular distribution of the sst2A receptor immunofluorescence in the human cerebral cortex at GW 19. A–A″) Intensely labeled sst2A receptor-immunoreactive neurons (red in A′–A″) form chain-like clusters in the middle part of the cortical plate (CP). Note that long sst2A receptor-immunoreactive radial processes reach the pial surface (PS). B–B″) Receptor-immunolabeled cells and processes (red in B, B″) are closely apposed by vimentin-positive processes (green in B′B″), suggesting migration of sst2A-labeled cells on radial glia. C–C″) Sst2A receptor-immunoreactive processes (red in C, C″) are contacted by fibers that are immunoreactive for SRIF (green in D, D″) (arrowheads), the endogen ligand of the receptor. Scale bars: 20 µm.

    Techniques Used: Immunofluorescence, Labeling, Immunolabeling, Migration

    Immunofluorescence of the sst2A receptor in coronal sections through the rat neocortical wall between E14 and P5. A–A″′) At E14, the sst2A receptor immunoreactivity is detected in the preplate (PP). Receptor immunoreactivity is observed in cell bodies and basal processes perpendicular to the pial surface (A″′). B–B″′) At E16, intense receptor immunoreactivity is confined to neuronal cells located in the subplate/intermediate zone (SP/IZ; defined by arrowheads). Immunolabeling is located in cell bodies and small processes of closely packed and presumably migrating neurons (B″′). C–C″′) At E18, the sst2A receptor immunoreactivity is confined to cells in the intermediate zone but absent from the adjacent subplate (defined by arrowheads). D–D″′) At E21, sst2A receptor immunoreactivity is concentrated in the subventricular zone (SVZ) and the adjacent deep part of IZ. Immunoreactivity is apparent in cell bodies and radially oriented processes (D, D″′). E) At P5, the sst2A receptor immunoreactivity is diffusely distributed over the neuropil. The labeling intensity decreases towards the deep layers. At high magnification, receptor immunoreactivity appears diffusely distributed within the neuropil (E″′). A″′, B″′, C″′, D″′ and E″′ represent magnifications of boxed areas on A″, B″, C″, D″ and E″, respectively. CP/MZ, cortical plate/marginal zone; CP, cortical plate; MZ, marginal zone; VZ, ventricular zone; I–VI, cortical layers I to VI; WM, white matter. Scale bars: A–A″, B–B″, 50 µm; C–C″, D–D″, E–E″, 100 µm; A″′–E″′,10 µm.
    Figure Legend Snippet: Immunofluorescence of the sst2A receptor in coronal sections through the rat neocortical wall between E14 and P5. A–A″′) At E14, the sst2A receptor immunoreactivity is detected in the preplate (PP). Receptor immunoreactivity is observed in cell bodies and basal processes perpendicular to the pial surface (A″′). B–B″′) At E16, intense receptor immunoreactivity is confined to neuronal cells located in the subplate/intermediate zone (SP/IZ; defined by arrowheads). Immunolabeling is located in cell bodies and small processes of closely packed and presumably migrating neurons (B″′). C–C″′) At E18, the sst2A receptor immunoreactivity is confined to cells in the intermediate zone but absent from the adjacent subplate (defined by arrowheads). D–D″′) At E21, sst2A receptor immunoreactivity is concentrated in the subventricular zone (SVZ) and the adjacent deep part of IZ. Immunoreactivity is apparent in cell bodies and radially oriented processes (D, D″′). E) At P5, the sst2A receptor immunoreactivity is diffusely distributed over the neuropil. The labeling intensity decreases towards the deep layers. At high magnification, receptor immunoreactivity appears diffusely distributed within the neuropil (E″′). A″′, B″′, C″′, D″′ and E″′ represent magnifications of boxed areas on A″, B″, C″, D″ and E″, respectively. CP/MZ, cortical plate/marginal zone; CP, cortical plate; MZ, marginal zone; VZ, ventricular zone; I–VI, cortical layers I to VI; WM, white matter. Scale bars: A–A″, B–B″, 50 µm; C–C″, D–D″, E–E″, 100 µm; A″′–E″′,10 µm.

    Techniques Used: Immunofluorescence, Immunolabeling, Labeling

    Effect of sst2A receptor activation on in vitro granule cell migration. A) Representative image of an external granular layer (EGL) microexplant after 3 days in vitro in culture. The core of the explant and surrounding scattered migrating granules cells are labeled with DAPI (blue). Neuronal processes are labeled by neuronal class III β-tubulin immunoreactivity (green). B) In individual granule cells, sst2A receptor immunoreactivity is visible in both neuronal perikarya and processes (red). C) Illustration of sst2A receptor immunolabeling (red) in a β-tubulin-immunoreactive (green) axon. Note the sst2A-immunoreactive puncta in a growth cone structure (arrow). D, E) In comparison to control (D) the number of migrating granule cells is significantly increased in 100 nm octreotide-treated EGL (E) microexplants. The octreotide-induced granule cell migration increase is dose-dependent as revealed by quantitative analysis (F). *p
    Figure Legend Snippet: Effect of sst2A receptor activation on in vitro granule cell migration. A) Representative image of an external granular layer (EGL) microexplant after 3 days in vitro in culture. The core of the explant and surrounding scattered migrating granules cells are labeled with DAPI (blue). Neuronal processes are labeled by neuronal class III β-tubulin immunoreactivity (green). B) In individual granule cells, sst2A receptor immunoreactivity is visible in both neuronal perikarya and processes (red). C) Illustration of sst2A receptor immunolabeling (red) in a β-tubulin-immunoreactive (green) axon. Note the sst2A-immunoreactive puncta in a growth cone structure (arrow). D, E) In comparison to control (D) the number of migrating granule cells is significantly increased in 100 nm octreotide-treated EGL (E) microexplants. The octreotide-induced granule cell migration increase is dose-dependent as revealed by quantitative analysis (F). *p

    Techniques Used: Activation Assay, In Vitro, Migration, Labeling, Immunolabeling

    Subcellular localization of sst2A receptor immunoreactivity in neocortical cells at E16. A–C) Pre-embedding immunogold immunohistochemistry of the sst2A receptor in the developing cortex at E16 demonstrates localization of immunoparticles at the internal surface of the plasma membrane (arrowheads). D,E) High magnification confocal microscopic analysis reveals agonist-induced redistribution of surface receptors to intracellular compartments. In control conditions, sst2A receptor immunoreactivity outlines the periphery of cells (D). Forty minutes after agonist administration, accumulation of immunoreactive puncta in the cytoplasm become evident (E). Scale bars, A, C, 500 nm; B, 1 µm; D, E, 10 µm.
    Figure Legend Snippet: Subcellular localization of sst2A receptor immunoreactivity in neocortical cells at E16. A–C) Pre-embedding immunogold immunohistochemistry of the sst2A receptor in the developing cortex at E16 demonstrates localization of immunoparticles at the internal surface of the plasma membrane (arrowheads). D,E) High magnification confocal microscopic analysis reveals agonist-induced redistribution of surface receptors to intracellular compartments. In control conditions, sst2A receptor immunoreactivity outlines the periphery of cells (D). Forty minutes after agonist administration, accumulation of immunoreactive puncta in the cytoplasm become evident (E). Scale bars, A, C, 500 nm; B, 1 µm; D, E, 10 µm.

    Techniques Used: Immunohistochemistry

    Regional, cellular and subcellular localization of sst2A receptor immunoreactivity on sagittal (A–H) and coronal (I–K) sections of the rat mesencephalon and diencephalon between E14 and E18. A) At E16, intense sst2A receptor immunoreactivity is observed in the substantia nigra (SN; boxed area) and along the medial forebrain bundle (mfb; arrowhead). B–B″) At E16, the sst2A receptor (red in B, B″) and tyrosine hydroxylase (TH) (green in B′, B″) immunoreactivities extensively overlap both in the SN and in emerging processes of the mfb. C–C″) At E18, sst2A receptor immunoreactivity is dramatically decreased in both the SN and the mfb. D–D″) High magnification microscopic images illustrate numerous sst2A receptor-immunoreactive fibers (red in D,D′) in the mfb at E16. Some of them are TH-positive (green in D′,D″) (arrowheads). E–E″) Some sst2A receptor-immunolabeled axons (red in E, E″) of the mfb express 5-HT (green in E′, E″) (arrowheads). F,G) Pre-embedding immunogold immunohistochemistry of the sst2A receptor in the mfb at E16 illustrates very high density of immunoparticles in axons (F) and growth cone-like structures (G). Note that although the majority of immunoparticles are intracellular, some are found associated to the plasma membrane. H) At E14, intense sst2A receptor immunolabeling is observed on sagittal sections in the developing hypothalamus (boxed area). I) Illustration of receptor immunoreactivity on coronal section at the level of hypothalamic area at E16. Note the receptor immunoreactivity in the caudal ganglionic eminence (CGE) (arrow). J,K) J represents magnification of boxed area in I. At high magnification, sst2A receptor immunoreactivity is found at the periphery of numerous hypothalamic neurons. III, third ventricle; HA, hypothalamic area. Scale bars: A, 500 µm; B–B″, C–C″, 50 µm; D–D″, E–E″, J, 20 µm; F, G, 1 µm; H, 250 µm; I, 200 µm; K, 10 µm.
    Figure Legend Snippet: Regional, cellular and subcellular localization of sst2A receptor immunoreactivity on sagittal (A–H) and coronal (I–K) sections of the rat mesencephalon and diencephalon between E14 and E18. A) At E16, intense sst2A receptor immunoreactivity is observed in the substantia nigra (SN; boxed area) and along the medial forebrain bundle (mfb; arrowhead). B–B″) At E16, the sst2A receptor (red in B, B″) and tyrosine hydroxylase (TH) (green in B′, B″) immunoreactivities extensively overlap both in the SN and in emerging processes of the mfb. C–C″) At E18, sst2A receptor immunoreactivity is dramatically decreased in both the SN and the mfb. D–D″) High magnification microscopic images illustrate numerous sst2A receptor-immunoreactive fibers (red in D,D′) in the mfb at E16. Some of them are TH-positive (green in D′,D″) (arrowheads). E–E″) Some sst2A receptor-immunolabeled axons (red in E, E″) of the mfb express 5-HT (green in E′, E″) (arrowheads). F,G) Pre-embedding immunogold immunohistochemistry of the sst2A receptor in the mfb at E16 illustrates very high density of immunoparticles in axons (F) and growth cone-like structures (G). Note that although the majority of immunoparticles are intracellular, some are found associated to the plasma membrane. H) At E14, intense sst2A receptor immunolabeling is observed on sagittal sections in the developing hypothalamus (boxed area). I) Illustration of receptor immunoreactivity on coronal section at the level of hypothalamic area at E16. Note the receptor immunoreactivity in the caudal ganglionic eminence (CGE) (arrow). J,K) J represents magnification of boxed area in I. At high magnification, sst2A receptor immunoreactivity is found at the periphery of numerous hypothalamic neurons. III, third ventricle; HA, hypothalamic area. Scale bars: A, 500 µm; B–B″, C–C″, 50 µm; D–D″, E–E″, J, 20 µm; F, G, 1 µm; H, 250 µm; I, 200 µm; K, 10 µm.

    Techniques Used: Immunolabeling, Immunohistochemistry

    Immunofluorescence of sst2A receptor in the rat perinatal rostral migratory stream. A–A″) In sagittal sections at P0, an intense band of sst2A receptor immunoreactivity is observed from the anterior subventricular zone (SVZa), through the rostral migratory stream (RMS) and ending in the olfactory bulb (OB). From the SVZa, shown in detail in the high magnification insets, chains of immunoreactive neurons perpendicular to the SVZa long axis extend into the white matter of the overlying cerebral cortex. B–B″ represents high magnification of the area labeled with asterisk in A. The sst2A receptor immunoreactive cells are principally localized along the ventral and dorsal surface of RMS. C) High magnification of sst2A receptor immunoreactivity at the entrance of RMS into the olfactory bulb at P5 illustrates immunoreactive cells at the surface of the stream as well as embedded in central position. D) In the dorsal part of the RMS, sst2A receptor-immunoreactive cells (red) contain NeuN labeling in their nuclei (green; arrowhead). E) Sst2A receptor- (red) and NeuN- (blue) double-labeled cells (arrowhead) of the RMS do not contain BrdU immunoreactivity (green), demonstrating that receptor expression is restricted to post-mitotic neurons. Scale bars: A–A″, 500 µm; B–B″, C, 100 µm; D, 20 µm; E, 10 µm.
    Figure Legend Snippet: Immunofluorescence of sst2A receptor in the rat perinatal rostral migratory stream. A–A″) In sagittal sections at P0, an intense band of sst2A receptor immunoreactivity is observed from the anterior subventricular zone (SVZa), through the rostral migratory stream (RMS) and ending in the olfactory bulb (OB). From the SVZa, shown in detail in the high magnification insets, chains of immunoreactive neurons perpendicular to the SVZa long axis extend into the white matter of the overlying cerebral cortex. B–B″ represents high magnification of the area labeled with asterisk in A. The sst2A receptor immunoreactive cells are principally localized along the ventral and dorsal surface of RMS. C) High magnification of sst2A receptor immunoreactivity at the entrance of RMS into the olfactory bulb at P5 illustrates immunoreactive cells at the surface of the stream as well as embedded in central position. D) In the dorsal part of the RMS, sst2A receptor-immunoreactive cells (red) contain NeuN labeling in their nuclei (green; arrowhead). E) Sst2A receptor- (red) and NeuN- (blue) double-labeled cells (arrowhead) of the RMS do not contain BrdU immunoreactivity (green), demonstrating that receptor expression is restricted to post-mitotic neurons. Scale bars: A–A″, 500 µm; B–B″, C, 100 µm; D, 20 µm; E, 10 µm.

    Techniques Used: Immunofluorescence, Labeling, Expressing

    Regional and cellular distribution of sst2A receptor immunoreactivity in the rat developing locus coeruleus. (A) At E18, strong sst2A receptor immunoreactivity is found not only in the rhombic lip (rl) and external granular layer (EGL) but also in the rostro-ventral part of the cerebellum between the cerebellar ventricular area (IV) and the ventral hindbrain (boxed area). B–B″) The large, elongated sst2A receptor-immunoreactive cells (red in B, B″) lie parallel with the ventricular surface. These neurons also express tyrosine hydroxylase (TH; green in B′, B″), a marker of catecholaminergic neurons. C) At E21, intense sst2A receptor immunoreactivity (red in C, C″) is observed in the developing locus coeruleus (LC) and overlap with TH immunolabeling (green in D′,D″). D represents high magnification of boxed area in C. Note that intense sst2A receptor immunoreactivity (red) outlines the periphery of TH-positive (green) neurons (arrowheads). E) At P3, the locus coeruleus exhibits also strong sst2A receptor immunoreactivity (red). The blue labeling represents DAPI staining. CB, cerebellum; IV, fourth ventricle. Scale bars: A, 200 µm; B–B″, C–C″, E, 100 µm; D–D″, 20 µm.
    Figure Legend Snippet: Regional and cellular distribution of sst2A receptor immunoreactivity in the rat developing locus coeruleus. (A) At E18, strong sst2A receptor immunoreactivity is found not only in the rhombic lip (rl) and external granular layer (EGL) but also in the rostro-ventral part of the cerebellum between the cerebellar ventricular area (IV) and the ventral hindbrain (boxed area). B–B″) The large, elongated sst2A receptor-immunoreactive cells (red in B, B″) lie parallel with the ventricular surface. These neurons also express tyrosine hydroxylase (TH; green in B′, B″), a marker of catecholaminergic neurons. C) At E21, intense sst2A receptor immunoreactivity (red in C, C″) is observed in the developing locus coeruleus (LC) and overlap with TH immunolabeling (green in D′,D″). D represents high magnification of boxed area in C. Note that intense sst2A receptor immunoreactivity (red) outlines the periphery of TH-positive (green) neurons (arrowheads). E) At P3, the locus coeruleus exhibits also strong sst2A receptor immunoreactivity (red). The blue labeling represents DAPI staining. CB, cerebellum; IV, fourth ventricle. Scale bars: A, 200 µm; B–B″, C–C″, E, 100 µm; D–D″, 20 µm.

    Techniques Used: Marker, Immunolabeling, Labeling, Staining

    Regional and cellular distribution of sst2A receptor immunoreactivity on coronal sections of the prenatal human cerebellum. A) At GW 19, intense receptor immunoreactivity (red in A, A″) is observed in the deep part of the external granular layer (EGL). Note the large number of DAPI-positive cell nuclei (blue in A′, A″) in the superficial EGL. B–B″′ represent high magnification of boxed area in A. The sst2A receptor immunoreactivity (red in B, B″′) is mainly distributed in the deep part of the EGL whereas SRIF-immunoreactive cells (green in B′, B″′) are mainly located in the deep part of the molecular layer (ML). C–C″′) At GW 20, the high density sst2A receptor immunoreactivity (red in C, C″′) in the deep EGL is still present. In addition, intense receptor immunolabeling is detected in the internal granular layer (IGL; asterisk) and overlap with NeuN-immunoreactive cells (green in C′–C″′). D represents magnification of boxed area in C. The sst2A receptor is expressed in cells bodies located in the deep part of the EGL. E–E″′) In the IGL, the vast majority of NeuN- (green in E′, E″′) and DAPI- (blue in E″, E″′) positive cell nuclei are outlined by sst2A receptor immunoreactivity (red in E, E″′) (arrowheads), suggesting that the receptor is expressed by migrating granule cells. Scale bars: A–A″, C–C″′, 100 µm; B–B″′, D, E–E″′, 20 µm.
    Figure Legend Snippet: Regional and cellular distribution of sst2A receptor immunoreactivity on coronal sections of the prenatal human cerebellum. A) At GW 19, intense receptor immunoreactivity (red in A, A″) is observed in the deep part of the external granular layer (EGL). Note the large number of DAPI-positive cell nuclei (blue in A′, A″) in the superficial EGL. B–B″′ represent high magnification of boxed area in A. The sst2A receptor immunoreactivity (red in B, B″′) is mainly distributed in the deep part of the EGL whereas SRIF-immunoreactive cells (green in B′, B″′) are mainly located in the deep part of the molecular layer (ML). C–C″′) At GW 20, the high density sst2A receptor immunoreactivity (red in C, C″′) in the deep EGL is still present. In addition, intense receptor immunolabeling is detected in the internal granular layer (IGL; asterisk) and overlap with NeuN-immunoreactive cells (green in C′–C″′). D represents magnification of boxed area in C. The sst2A receptor is expressed in cells bodies located in the deep part of the EGL. E–E″′) In the IGL, the vast majority of NeuN- (green in E′, E″′) and DAPI- (blue in E″, E″′) positive cell nuclei are outlined by sst2A receptor immunoreactivity (red in E, E″′) (arrowheads), suggesting that the receptor is expressed by migrating granule cells. Scale bars: A–A″, C–C″′, 100 µm; B–B″′, D, E–E″′, 20 µm.

    Techniques Used: Immunolabeling

    Regional, cellular and subcellular distribution of sst2A receptor immunoreactivity in sagittal sections of the rat cerebellum during pre- and postnatal development. A) At E14, sst2A receptor immunoreactivity is detected in the developing cerebellum (boxed area). Note the strong expression of the receptor in the developing hypothalamus (arrowhead) and rhombencephalon (asterisk). B) The sst2A receptor immunoreactivity is intense at the outer border of the cerebellar neuroepithelium (asterisk) and the adjacent upper component of the rhombic lip (rl). C) At E16, strong cellular sst2A receptor labeling is evident in the dorsal part of the cerebellum, where the progenitors of the external granular layer (EGL) migrate. D,E) Pre-embedding immunogold immunohistochemistry of the sst2A receptor in the developing external germinal layer at E16 illustrates that immunoparticles are predominantly localized at the internal surface of the plasma membrane (arrowheads). F,G) High magnification confocal microscopic analysis of the developing EGL reveals redistribution of surface receptors to intracellular compartments upon agonist stimulation. In control conditions, sst2A receptor immunoreactivity outlines the periphery of neurons (F). Forty minutes after agonist administration, accumulation of immunoreactive puncta in the cytoplasm become evident (G). H) At P5, intense sst2A receptor immunofluorescence is observed in the EGL. I–I″ represent magnification of boxed area in H. The sst2A receptor-immunoreactive neurons (red) are predominantly located in the deep part of EGL (I). The Ki-67-immuonreactive proliferative neurons (green) are distributed predominantly in the superficial EGL (I′). Accordingly only few sst2A receptor-immunoreactive neurons are Ki-67-positive (I″; arrowheads). J–J″) In the EGL, most sst2A receptor-immunolabeled neurons (red in J, J″) are positive for the neuronal-specific nuclear protein NeuN (green in J′, J″) (arrowheads) and demonstrate the post-mitotic nature of sst2A-positive EGL neurons. K, K″) At P5, the large unipolar calretinin-immunoreactive brush cells (green in K′, K″) are sst2A receptor immunoreactive (red in K, K″). Note the colocalization of sst2A receptor and calretinin in a long brush cell process (arrowhead). cb, cerebellar neuroepithelium; CB, cerebellum. Scale bars: A, 250 µm; B, H, K–K″, 50 µm; C, 20 µm, D, 200 nm; E, 400 nm. F, G, I–I″, J–J″, 10 µm.
    Figure Legend Snippet: Regional, cellular and subcellular distribution of sst2A receptor immunoreactivity in sagittal sections of the rat cerebellum during pre- and postnatal development. A) At E14, sst2A receptor immunoreactivity is detected in the developing cerebellum (boxed area). Note the strong expression of the receptor in the developing hypothalamus (arrowhead) and rhombencephalon (asterisk). B) The sst2A receptor immunoreactivity is intense at the outer border of the cerebellar neuroepithelium (asterisk) and the adjacent upper component of the rhombic lip (rl). C) At E16, strong cellular sst2A receptor labeling is evident in the dorsal part of the cerebellum, where the progenitors of the external granular layer (EGL) migrate. D,E) Pre-embedding immunogold immunohistochemistry of the sst2A receptor in the developing external germinal layer at E16 illustrates that immunoparticles are predominantly localized at the internal surface of the plasma membrane (arrowheads). F,G) High magnification confocal microscopic analysis of the developing EGL reveals redistribution of surface receptors to intracellular compartments upon agonist stimulation. In control conditions, sst2A receptor immunoreactivity outlines the periphery of neurons (F). Forty minutes after agonist administration, accumulation of immunoreactive puncta in the cytoplasm become evident (G). H) At P5, intense sst2A receptor immunofluorescence is observed in the EGL. I–I″ represent magnification of boxed area in H. The sst2A receptor-immunoreactive neurons (red) are predominantly located in the deep part of EGL (I). The Ki-67-immuonreactive proliferative neurons (green) are distributed predominantly in the superficial EGL (I′). Accordingly only few sst2A receptor-immunoreactive neurons are Ki-67-positive (I″; arrowheads). J–J″) In the EGL, most sst2A receptor-immunolabeled neurons (red in J, J″) are positive for the neuronal-specific nuclear protein NeuN (green in J′, J″) (arrowheads) and demonstrate the post-mitotic nature of sst2A-positive EGL neurons. K, K″) At P5, the large unipolar calretinin-immunoreactive brush cells (green in K′, K″) are sst2A receptor immunoreactive (red in K, K″). Note the colocalization of sst2A receptor and calretinin in a long brush cell process (arrowhead). cb, cerebellar neuroepithelium; CB, cerebellum. Scale bars: A, 250 µm; B, H, K–K″, 50 µm; C, 20 µm, D, 200 nm; E, 400 nm. F, G, I–I″, J–J″, 10 µm.

    Techniques Used: Expressing, Labeling, Immunohistochemistry, Immunofluorescence, Immunolabeling

    Effect of sst2A receptor agonist on axonal and dendritic patterning. A–A″) Representative image of sst2A receptor localization in a primary hippocampal cell after 24 h in vitro in culture. Receptor immunoreactivity (green in A, A″) is present in the cell body and processes. Cell morphology is revealed by actin-binding protein phalloidin (red in A′, A″). Note that sst2A receptor immunoreactivity is also present in growth cones (insets in A–A″). B–D) Representative images of neurons from control (ctrl; B), 10 nM octreotide-treated (10 nM oct.; C) and 50 nM octreotide-treated (50 nM oct.; D) cultures. Arrows depict the axonal process which appears longer when cells are treated with 50 nM octreotide. E) Quantitative analysis reveals that the axon length (right panel) is significantly increased in the 50 nM oct. group when compared to the control group. The mean cell body surface (left panel) and the mean dendritic length (middle panel) are not modified by sst2A receptor agonist treatments. Values (mean±SEM) are expressed in relation to an arbitrary unit (100%) of the control values. *p
    Figure Legend Snippet: Effect of sst2A receptor agonist on axonal and dendritic patterning. A–A″) Representative image of sst2A receptor localization in a primary hippocampal cell after 24 h in vitro in culture. Receptor immunoreactivity (green in A, A″) is present in the cell body and processes. Cell morphology is revealed by actin-binding protein phalloidin (red in A′, A″). Note that sst2A receptor immunoreactivity is also present in growth cones (insets in A–A″). B–D) Representative images of neurons from control (ctrl; B), 10 nM octreotide-treated (10 nM oct.; C) and 50 nM octreotide-treated (50 nM oct.; D) cultures. Arrows depict the axonal process which appears longer when cells are treated with 50 nM octreotide. E) Quantitative analysis reveals that the axon length (right panel) is significantly increased in the 50 nM oct. group when compared to the control group. The mean cell body surface (left panel) and the mean dendritic length (middle panel) are not modified by sst2A receptor agonist treatments. Values (mean±SEM) are expressed in relation to an arbitrary unit (100%) of the control values. *p

    Techniques Used: In Vitro, Binding Assay, Modification

    5) Product Images from "Bacteria-Induced Uroplakin Signaling Mediates Bladder Response to Infection"

    Article Title: Bacteria-Induced Uroplakin Signaling Mediates Bladder Response to Infection

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1000415

    Co-localization of uroplakins and FimH binding sites on PD07i cell surface. The surface-expressed uroplakins on PD07i cells were detected using antisera against individual uroplakins Ia (B), Ib (E), II (H), and IIIa (K), followed by Alexa Fluor 594-conjugated donkey anti-rabbit IgG; while FimH was localized using biotinylated FimH/C complex, followed with FITC-conjugated streptavidin (A, D, G, J). Arrows mark the co-localization of surface-expressed uroplakins and FimH binding sties (A–L).
    Figure Legend Snippet: Co-localization of uroplakins and FimH binding sites on PD07i cell surface. The surface-expressed uroplakins on PD07i cells were detected using antisera against individual uroplakins Ia (B), Ib (E), II (H), and IIIa (K), followed by Alexa Fluor 594-conjugated donkey anti-rabbit IgG; while FimH was localized using biotinylated FimH/C complex, followed with FITC-conjugated streptavidin (A, D, G, J). Arrows mark the co-localization of surface-expressed uroplakins and FimH binding sties (A–L).

    Techniques Used: Binding Assay, IA

    6) Product Images from "Autocrine laminin-5 ligates ?6?4 integrin and activates RAC and NF?B to mediate anchorage-independent survival of mammary tumors"

    Article Title: Autocrine laminin-5 ligates ?6?4 integrin and activates RAC and NF?B to mediate anchorage-independent survival of mammary tumors

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200302023

    NF κ B activation is necessary and sufficient for anchorage-independent survival of MECs. (A and B) Cell viability was calculated using the Live/Dead assay for T4-2s (A) and T4 β4Δ cyto/V12RAC (B) MECs treated with either vehicle (Control), a peptide that inhibits nuclear translocation of NFκB SN50 (SN50), or a nonfunction-blocking peptide SN50M (SN50M) grown in rBM for 96 h with or without a function-blocking mAb to β1 integrin. (C) Cell viability calculated using the Live/Dead assay for T4-2 controls (T4-2) or T4-2s expressing a mutant IκBα (IκBαM) grown and treated as in A. (D) Confocal immunofluorescence microscopy images of Cytokeratin 18 (Texas red) and NFκB p65 (FITC) in S-1 controls (S-1) and S-1s overexpressing an exogenous NFκB p65 (S-1 p65) showing constitutive nuclear NFκB p65 in the S-1 p65 structures (arrows, nuclei as indicated by “n”). Bar, 20 μm. (E) Quantification of 100–200 representative cells assayed from images similar to D demonstrating a significant increase in nuclear NFκB p65 in S-1s overexpressing NFκB p65 (S-1 p65). (F) Cell viability was calculated using the Live/Dead assay for S-1 and S-1 cells overexpressing NFκB p65 (S-1 p65) grown and treated as described for A. (G) Soft agar assay results demonstrating overexpressing exogenous NFkB p65 (S-1 p65) permits S-1s (S-1) to form colonies in soft agar. Results for A–C and E–G are the mean ± SEM of three to five experiments. *, P ≤ 0.05; ***, P ≤ 0.001.
    Figure Legend Snippet: NF κ B activation is necessary and sufficient for anchorage-independent survival of MECs. (A and B) Cell viability was calculated using the Live/Dead assay for T4-2s (A) and T4 β4Δ cyto/V12RAC (B) MECs treated with either vehicle (Control), a peptide that inhibits nuclear translocation of NFκB SN50 (SN50), or a nonfunction-blocking peptide SN50M (SN50M) grown in rBM for 96 h with or without a function-blocking mAb to β1 integrin. (C) Cell viability calculated using the Live/Dead assay for T4-2 controls (T4-2) or T4-2s expressing a mutant IκBα (IκBαM) grown and treated as in A. (D) Confocal immunofluorescence microscopy images of Cytokeratin 18 (Texas red) and NFκB p65 (FITC) in S-1 controls (S-1) and S-1s overexpressing an exogenous NFκB p65 (S-1 p65) showing constitutive nuclear NFκB p65 in the S-1 p65 structures (arrows, nuclei as indicated by “n”). Bar, 20 μm. (E) Quantification of 100–200 representative cells assayed from images similar to D demonstrating a significant increase in nuclear NFκB p65 in S-1s overexpressing NFκB p65 (S-1 p65). (F) Cell viability was calculated using the Live/Dead assay for S-1 and S-1 cells overexpressing NFκB p65 (S-1 p65) grown and treated as described for A. (G) Soft agar assay results demonstrating overexpressing exogenous NFkB p65 (S-1 p65) permits S-1s (S-1) to form colonies in soft agar. Results for A–C and E–G are the mean ± SEM of three to five experiments. *, P ≤ 0.05; ***, P ≤ 0.001.

    Techniques Used: Activation Assay, Live Dead Assay, Translocation Assay, Blocking Assay, Expressing, Mutagenesis, Immunofluorescence, Microscopy, Soft Agar Assay

    7) Product Images from "Effect of wild bitter gourd treatment on inflammatory responses in BALB/c mice with sepsis"

    Article Title: Effect of wild bitter gourd treatment on inflammatory responses in BALB/c mice with sepsis

    Journal: Biomedicine

    doi: 10.7603/s40681-014-0017-y

    COX-2, NF-κB, and iNOS inflammatory protein expression in six groups of mice after four-week test diet, values mean ± SD of each group. Values not sharing a superscript (a-e) differ significantly by one-way ANOVA and Duncan’s Multiple Range Test ( p
    Figure Legend Snippet: COX-2, NF-κB, and iNOS inflammatory protein expression in six groups of mice after four-week test diet, values mean ± SD of each group. Values not sharing a superscript (a-e) differ significantly by one-way ANOVA and Duncan’s Multiple Range Test ( p

    Techniques Used: Expressing, Mouse Assay

    8) Product Images from "Genetically modified human placenta-derived mesenchymal stem cells with FGF-2 and PDGF-BB enhance neovascularization in a model of hindlimb ischemia"

    Article Title: Genetically modified human placenta-derived mesenchymal stem cells with FGF-2 and PDGF-BB enhance neovascularization in a model of hindlimb ischemia

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2015.4089

    hPDMSC survival in the ischemic tissue. Immunohistochemical staining with an antibody against human-specific surface of intact mitochondria protein indicated that xenografted hPDMSCs survived in the ischemic tissue for at least four weeks. Scale bar, 50 µ m; magnification ×3. hPDMSC, human placenta-derived mesenchymal stem cell. AD-F-P, adenoviral bicistronic vector containing FGF2 and PDGF-BB; ctrl, control without PDMSCs.
    Figure Legend Snippet: hPDMSC survival in the ischemic tissue. Immunohistochemical staining with an antibody against human-specific surface of intact mitochondria protein indicated that xenografted hPDMSCs survived in the ischemic tissue for at least four weeks. Scale bar, 50 µ m; magnification ×3. hPDMSC, human placenta-derived mesenchymal stem cell. AD-F-P, adenoviral bicistronic vector containing FGF2 and PDGF-BB; ctrl, control without PDMSCs.

    Techniques Used: Immunohistochemistry, Staining, Derivative Assay, Plasmid Preparation

    9) Product Images from "Secreted multifunctional Glyceraldehyde-3-phosphate dehydrogenase sequesters lactoferrin and iron into cells via a non-canonical pathway"

    Article Title: Secreted multifunctional Glyceraldehyde-3-phosphate dehydrogenase sequesters lactoferrin and iron into cells via a non-canonical pathway

    Journal: Scientific Reports

    doi: 10.1038/srep18465

    Co-trafficking and interaction between GAPDH and Lf in cells ( a ) Lf and sGAPDH signals are co-localized inside cells. ( b ) (i) Immunogold labeling transmission electron microscopy of purified endosomes from J774 cells demonstrates the co-localization of lactoferrin (20 nm particles indicated with arrowheads) and monoclonal anti GAPDH antibody (5 nm particles marked with arrows). (ii) In place of specific antibody isotype control antibody (5 nm particle) was used. ( c ) Intracellular interaction of Lf and GAPDH was demonstrated by acceptor photobleaching FRET. Bleaching of Lf-TRITC or GAPDH-TRITC (acceptor) signal is accompanied by an increase in the GAPDH-FITC or Lf-FITC (donor) signal in J774 or CHO-TRVb cells respectively (arrows). ( d ) FRET efficiency was calculated from 25 cells in both cases. FRET efficiency was calculated using the following formula, FRET efficienc = [Donor intensity (post-bleach)-Donor intensity (pre-bleach)]/Donor intensity (post-bleach). In control experiments the FRET donor replaced by IgG-FITC. FRET experiments were conducted using a Nikon A1R confocal microscope. ( e ) Interaction between internalized GAPDH and Lf was fu rther confirmed by co-immunoprecipitation from cytosolic fraction of J774 cells. Lf along with biotinylated GAPDH (to distinguish from intracellular GAPDH) was allowed to internalize into J774 cells for 1 hr at 37 °C, cells were then washed and treated with pronase. Lf was immunoprecepitated (IP) using anti-Lf antibody coupled magnabeads. Co-immunoprecipitated biotinylated GAPDH was then detected after immunoblotting (IB) with streptavidin-HRP. Control was run in parallel wherein the cytoplasmic fraction was incubated with isotype IgG coupled magnabeads.
    Figure Legend Snippet: Co-trafficking and interaction between GAPDH and Lf in cells ( a ) Lf and sGAPDH signals are co-localized inside cells. ( b ) (i) Immunogold labeling transmission electron microscopy of purified endosomes from J774 cells demonstrates the co-localization of lactoferrin (20 nm particles indicated with arrowheads) and monoclonal anti GAPDH antibody (5 nm particles marked with arrows). (ii) In place of specific antibody isotype control antibody (5 nm particle) was used. ( c ) Intracellular interaction of Lf and GAPDH was demonstrated by acceptor photobleaching FRET. Bleaching of Lf-TRITC or GAPDH-TRITC (acceptor) signal is accompanied by an increase in the GAPDH-FITC or Lf-FITC (donor) signal in J774 or CHO-TRVb cells respectively (arrows). ( d ) FRET efficiency was calculated from 25 cells in both cases. FRET efficiency was calculated using the following formula, FRET efficienc = [Donor intensity (post-bleach)-Donor intensity (pre-bleach)]/Donor intensity (post-bleach). In control experiments the FRET donor replaced by IgG-FITC. FRET experiments were conducted using a Nikon A1R confocal microscope. ( e ) Interaction between internalized GAPDH and Lf was fu rther confirmed by co-immunoprecipitation from cytosolic fraction of J774 cells. Lf along with biotinylated GAPDH (to distinguish from intracellular GAPDH) was allowed to internalize into J774 cells for 1 hr at 37 °C, cells were then washed and treated with pronase. Lf was immunoprecepitated (IP) using anti-Lf antibody coupled magnabeads. Co-immunoprecipitated biotinylated GAPDH was then detected after immunoblotting (IB) with streptavidin-HRP. Control was run in parallel wherein the cytoplasmic fraction was incubated with isotype IgG coupled magnabeads.

    Techniques Used: Labeling, Transmission Assay, Electron Microscopy, Purification, Microscopy, Immunoprecipitation, Incubation

    10) Product Images from "Limited role of regulatory T cells during acute Theiler virus-induced encephalitis in resistant C57BL/6 mice"

    Article Title: Limited role of regulatory T cells during acute Theiler virus-induced encephalitis in resistant C57BL/6 mice

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-014-0180-9

    Early recruitment of T cells into Theiler’s murine encephalomyelitis virus (TMEV)-infected brain in the absence of Tregs. Following intraperitoneal administration of PBS or diphtheria toxin (DT), DEREG mice were intracerebrally infected with TMEV. (A) Immunohistochemistry of TMEV infected brains at 3 days post inoculation (dpi) (left panel) and 7 dpi (right panel) reveals higher numbers of CD3 + T cells only at 3 dpi in DT-treated mice (lower panel) compared to PBS-treated mice (upper panel). (B) Quantification of CD3 + T cells in the cerebral neuroparenchyma of 6 to 8 infected mice reveals a significantly increased number on T cells in DT-treated mice at 3 dpi. Box and whisker plots display median and quartiles with maximum and minimum values. * P -value
    Figure Legend Snippet: Early recruitment of T cells into Theiler’s murine encephalomyelitis virus (TMEV)-infected brain in the absence of Tregs. Following intraperitoneal administration of PBS or diphtheria toxin (DT), DEREG mice were intracerebrally infected with TMEV. (A) Immunohistochemistry of TMEV infected brains at 3 days post inoculation (dpi) (left panel) and 7 dpi (right panel) reveals higher numbers of CD3 + T cells only at 3 dpi in DT-treated mice (lower panel) compared to PBS-treated mice (upper panel). (B) Quantification of CD3 + T cells in the cerebral neuroparenchyma of 6 to 8 infected mice reveals a significantly increased number on T cells in DT-treated mice at 3 dpi. Box and whisker plots display median and quartiles with maximum and minimum values. * P -value

    Techniques Used: Infection, Mouse Assay, Immunohistochemistry, Whisker Assay

    11) Product Images from "Multiplex giant magnetoresistive biosensor microarrays identify interferon-associated autoantibodies in systemic lupus erythematosus"

    Article Title: Multiplex giant magnetoresistive biosensor microarrays identify interferon-associated autoantibodies in systemic lupus erythematosus

    Journal: Scientific Reports

    doi: 10.1038/srep27623

    GMR biosensor autoantigen microarrays. ( a ) Optical images of a GMR biosensor chip and a cartridge with a reaction well (left). The sensor chip measures 10 × 12 mm and consists of an array of 8 × 10 sensors (total 80 sensors). Each sensor size is 100 × 100 μm (right). ( b ) A schematic of assaying antibody reactivity to autoantigens (not to scale). (1) Autoantigens were printed on the surface of the chip’s sensors. (2) The sample was added to the reaction well, allowing antibodies to bind to their corresponding antigens. (3) After washing, species-specific, biotinylated anti-IgG antibodies were used as a secondary reagent. (4) Streptavidin-coated MNPs bind to the biotinylated detection antibodies, and the respective sensor detects stray field from the bound MNPs.
    Figure Legend Snippet: GMR biosensor autoantigen microarrays. ( a ) Optical images of a GMR biosensor chip and a cartridge with a reaction well (left). The sensor chip measures 10 × 12 mm and consists of an array of 8 × 10 sensors (total 80 sensors). Each sensor size is 100 × 100 μm (right). ( b ) A schematic of assaying antibody reactivity to autoantigens (not to scale). (1) Autoantigens were printed on the surface of the chip’s sensors. (2) The sample was added to the reaction well, allowing antibodies to bind to their corresponding antigens. (3) After washing, species-specific, biotinylated anti-IgG antibodies were used as a secondary reagent. (4) Streptavidin-coated MNPs bind to the biotinylated detection antibodies, and the respective sensor detects stray field from the bound MNPs.

    Techniques Used: Chromatin Immunoprecipitation

    12) Product Images from "Characterisation of the bovine enteric calici‐like virus, Newbury agent 1"

    Article Title: Characterisation of the bovine enteric calici‐like virus, Newbury agent 1

    Journal: FEMS Microbiology Letters

    doi: 10.1111/j.1574-6968.2000.tb09370.x

    NA1 virus particles trapped by SPIEM from the day 2 faecal sample of gnotobiotic calf 1424 using a 1:20 dilution of the NA1 immunoglobulin concentrate. Bar indicates 100 nm. Inset shows surface structure of an NA1 particle and spiked outer edge; bar indicates 50 nm. Stained with 2% PTA at pH 6.0.
    Figure Legend Snippet: NA1 virus particles trapped by SPIEM from the day 2 faecal sample of gnotobiotic calf 1424 using a 1:20 dilution of the NA1 immunoglobulin concentrate. Bar indicates 100 nm. Inset shows surface structure of an NA1 particle and spiked outer edge; bar indicates 50 nm. Stained with 2% PTA at pH 6.0.

    Techniques Used: Staining

    Clinical signs and faecal NA1 excretion by SPIEM (white box) in three gnotobiotic calves inoculated with NA1. Faecal colour changes (box with dots), increased rectal temperatures (box with dashed lines) and diminished appetite (box with lines) are indicated. Absence of a parameter indicates it was not present. Asterisks indicate that a faecal sample could not be collected on that day. The diminished appetite and abnormal faecal colour lasted till day 7 p.i. for calf 1424 (not shown). Error bars represent standard deviations of the mean particle counts per field.
    Figure Legend Snippet: Clinical signs and faecal NA1 excretion by SPIEM (white box) in three gnotobiotic calves inoculated with NA1. Faecal colour changes (box with dots), increased rectal temperatures (box with dashed lines) and diminished appetite (box with lines) are indicated. Absence of a parameter indicates it was not present. Asterisks indicate that a faecal sample could not be collected on that day. The diminished appetite and abnormal faecal colour lasted till day 7 p.i. for calf 1424 (not shown). Error bars represent standard deviations of the mean particle counts per field.

    Techniques Used:

    Mean number of NA1 particles (•) trapped per field by SPIEM in fractions from a representative CsCl density gradient centrifuged to equilibrium. Buoyant density (◯) determined by refractometry.
    Figure Legend Snippet: Mean number of NA1 particles (•) trapped per field by SPIEM in fractions from a representative CsCl density gradient centrifuged to equilibrium. Buoyant density (◯) determined by refractometry.

    Techniques Used:

    (a) Western blotting analysis of fractions from day 0 (track 1) and day 2 (track 2) faecal samples of calf 1424 purified by CsCl centrifugation stained with the post‐inoculation NA1 serum. A mean of 130.5 NA1 particles per field were present by SPIEM in the fraction from the day 2 sample; no particles were detected in the fraction from the day 0 sample. Track 3: bovine IgG as a control for electroblotting and immunostaining. M, molecular mass markers (kDa). (b) Western blotting analysis of fractions from the day 2 faecal sample of calf 1424 stained with the pre‐ (track 2) and the post‐inoculation NA1 serum (track 4). Tracks 1 and 3: bovine IgG as a control for electroblotting and immunostaining. M, molecular mass markers (kDa).
    Figure Legend Snippet: (a) Western blotting analysis of fractions from day 0 (track 1) and day 2 (track 2) faecal samples of calf 1424 purified by CsCl centrifugation stained with the post‐inoculation NA1 serum. A mean of 130.5 NA1 particles per field were present by SPIEM in the fraction from the day 2 sample; no particles were detected in the fraction from the day 0 sample. Track 3: bovine IgG as a control for electroblotting and immunostaining. M, molecular mass markers (kDa). (b) Western blotting analysis of fractions from the day 2 faecal sample of calf 1424 stained with the pre‐ (track 2) and the post‐inoculation NA1 serum (track 4). Tracks 1 and 3: bovine IgG as a control for electroblotting and immunostaining. M, molecular mass markers (kDa).

    Techniques Used: Western Blot, Purification, Centrifugation, Staining, Immunostaining

    (a) Molecular mass estimation of the capsid protein of NA1 particles purified by CsCl density gradient centrifugation. M, molecular masses of biotinylated standard proteins (kDa). Track 1: bovine IgG as a control for blotting and staining with the anti‐bovine conjugate; track 2: biotinylated standard proteins; track 3: biotinylated standard proteins plus the NA1 capsid protein; track 4: the NA1 capsid protein; arrow indicates the NA1 protein. (b) Molecular mass estimation of the capsid protein of FCV grown in CRFK cells using the same electrophoresis conditions as those used for NA1. Arrow indicates the FCV capsid protein. M, molecular masses of biotinylated standard proteins (kDa). Track 1: preparation from uninfected CRFK cells; track 2: biotinylated standard proteins plus the preparation from FCV‐infected cells; track 3: FCV‐infected cells; track 4: rabbit IgG (the FCV NADC rabbit antiserum) used as a control for blotting and immunostaining with the anti‐rabbit conjugate.
    Figure Legend Snippet: (a) Molecular mass estimation of the capsid protein of NA1 particles purified by CsCl density gradient centrifugation. M, molecular masses of biotinylated standard proteins (kDa). Track 1: bovine IgG as a control for blotting and staining with the anti‐bovine conjugate; track 2: biotinylated standard proteins; track 3: biotinylated standard proteins plus the NA1 capsid protein; track 4: the NA1 capsid protein; arrow indicates the NA1 protein. (b) Molecular mass estimation of the capsid protein of FCV grown in CRFK cells using the same electrophoresis conditions as those used for NA1. Arrow indicates the FCV capsid protein. M, molecular masses of biotinylated standard proteins (kDa). Track 1: preparation from uninfected CRFK cells; track 2: biotinylated standard proteins plus the preparation from FCV‐infected cells; track 3: FCV‐infected cells; track 4: rabbit IgG (the FCV NADC rabbit antiserum) used as a control for blotting and immunostaining with the anti‐rabbit conjugate.

    Techniques Used: Purification, Gradient Centrifugation, Staining, Electrophoresis, Infection, Immunostaining

    13) Product Images from "Grafting of Striatal Precursor Cells into Hippocampus Shortly after Status Epilepticus Restrains Chronic Temporal Lobe Epilepsy"

    Article Title: Grafting of Striatal Precursor Cells into Hippocampus Shortly after Status Epilepticus Restrains Chronic Temporal Lobe Epilepsy

    Journal: Experimental neurology

    doi: 10.1016/j.expneurol.2008.04.040

    Differentiation of grafted striatal precursor cells into neurons and gamma-amino butyric acid (GABA) positive neurons. Figures A1–A3 show examples of 5′-bromodeoxyuridine (BrdU) positive grafted cells that differentiated into neuron-specific nuclear antigen (NeuN) positive neurons (arrows), using BrdU and NeuN dual immunofluorescence. Figures A4–A8 illustrate Z-section analyses of grafted cells that are positive both BrdU and NeuN, using confocal microscopy. Figures B1–B3 show examples of grafted cells that differentiated into GABA-positive neurons (arrows), using BrdU and GABA dual immunofluorescence. Figures B4–B8 illustrates Z-section analyses of a grafted cell that is positive for both BrdU and GABA using confocal microscopy. The bar chart in C1 depicts average numbers of BrdU positive cells, NeuN positive neurons, and GABA-ergic neurons derived from individual striatal precursor cell grafts in the hippocampus. A1–A3, B1–B3 = 20μm; A4–A8, B4–B8 = 10μm.
    Figure Legend Snippet: Differentiation of grafted striatal precursor cells into neurons and gamma-amino butyric acid (GABA) positive neurons. Figures A1–A3 show examples of 5′-bromodeoxyuridine (BrdU) positive grafted cells that differentiated into neuron-specific nuclear antigen (NeuN) positive neurons (arrows), using BrdU and NeuN dual immunofluorescence. Figures A4–A8 illustrate Z-section analyses of grafted cells that are positive both BrdU and NeuN, using confocal microscopy. Figures B1–B3 show examples of grafted cells that differentiated into GABA-positive neurons (arrows), using BrdU and GABA dual immunofluorescence. Figures B4–B8 illustrates Z-section analyses of a grafted cell that is positive for both BrdU and GABA using confocal microscopy. The bar chart in C1 depicts average numbers of BrdU positive cells, NeuN positive neurons, and GABA-ergic neurons derived from individual striatal precursor cell grafts in the hippocampus. A1–A3, B1–B3 = 20μm; A4–A8, B4–B8 = 10μm.

    Techniques Used: Immunofluorescence, Confocal Microscopy, Derivative Assay

    Differentiation of grafted striatal precursor cells into subclasses of gamma-amino butyric acid (GABA) positive neurons, visualized through dual immunofluorescence and confocal microscopy. The figures show examples of 5′-bromodeoxyuridine (BrdU) positive grafted cells that differentiate into interneurons positive for calbindin (A1–A3), parvalbumin (PV, B1–B3), calretinin (C1–C3) and neuropeptide Y (NPY, D1–D3). Figures A4–A8, B4–B8, C4–C8 and D4–D8 illustrate Z-section analyses of grafted cells that are positive BrdU calbindin, BrdU parvalbumin, BrdU calretinin, and BrdU neuropeptide Y respectively. The bar chart in E1 depicts average numbers of interneurons positive for calbindin, parvalbumin, calretinin and neuropeptide Y in individual striatal precursor cell grafts. Scale bar, A1–A3, B1–B3, C1–C3, D1–D3 = 10μm; A4–A8, B4–B8, C4–C8, D4–D8 = 5μm. CBN, calbindin, PV, parvalbumin, CR, calretinin, and NPY, neuropeptide Y.
    Figure Legend Snippet: Differentiation of grafted striatal precursor cells into subclasses of gamma-amino butyric acid (GABA) positive neurons, visualized through dual immunofluorescence and confocal microscopy. The figures show examples of 5′-bromodeoxyuridine (BrdU) positive grafted cells that differentiate into interneurons positive for calbindin (A1–A3), parvalbumin (PV, B1–B3), calretinin (C1–C3) and neuropeptide Y (NPY, D1–D3). Figures A4–A8, B4–B8, C4–C8 and D4–D8 illustrate Z-section analyses of grafted cells that are positive BrdU calbindin, BrdU parvalbumin, BrdU calretinin, and BrdU neuropeptide Y respectively. The bar chart in E1 depicts average numbers of interneurons positive for calbindin, parvalbumin, calretinin and neuropeptide Y in individual striatal precursor cell grafts. Scale bar, A1–A3, B1–B3, C1–C3, D1–D3 = 10μm; A4–A8, B4–B8, C4–C8, D4–D8 = 5μm. CBN, calbindin, PV, parvalbumin, CR, calretinin, and NPY, neuropeptide Y.

    Techniques Used: Immunofluorescence, Confocal Microscopy

    14) Product Images from "NeuroD2 controls inhibitory circuit formation in the molecular layer of the cerebellum"

    Article Title: NeuroD2 controls inhibitory circuit formation in the molecular layer of the cerebellum

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-37850-7

    Surviving NeuroD2-deficient granule cells terminally differentiate and functionally integrate into excitatory cerebellar circuits. ( A ) Granule cells in Neurod2 −/− mutants extend claw-like telodendria (arrow) and axons projecting towards the ML (arrowhead). Golgi impregnation of sagittal sections (P25). Scale bar, 20 µm. ( B ) Fluorescent immunostaining for GABA Aα6 receptor on sagittal sections (lobule 5) from Neurod2 −/− mutants and controls (P25). (Right panels) magnification of boxed area in left panels. Scale bar, 600 µm. ( C ) Left: Schematic of PF EPSC recordings from Purkinje cells. PFs were stimulated in the ML (stim) while Purkinje cells were held in whole-cell voltage clamp (rec). Right: Representative traces of PF EPSCs from controls and Neurod2 −/− mutants. Paired-pulses reveal similar EPSC2 facilitation compared to EPSC1. ( D ) Left: Schematic of recordings of CF EPSCs from Purkinje cells. CFs were stimulated in the GL (stim) while Purkinje cells were held in whole-cell voltage clamp (rec). Right: Representative traces of CF EPSCs from control and Neurod2 −/− mutants. Paired-pulses reveal similar depression of EPSC2 compared to EPSC1. ( E ) Normal number and distribution of PFs and their terminals in Neurod2 −/− mutants compared to controls. Fluorescent immunostaining for Purkinje cell dendrites (PV) and PFs (calretinin, CR; or VGLUT1) (age 1 year). Note differences in PV staining due to loss of PV + MLIs in Neurod2 −/− mutants. Scale bars, 20 µm. (F) (Upper panels) Electron microscopy of granule cells in Neurod2 −/− mutants exhibits a similar distribution of nuclear heterochromatin compared to controls (age 1 year). Scale bar, 1.7 µm. (Lower panels) Ultrastructure of PF/Purkinje cell synapses (arrowheads) in Neurod2 −/− mutants compared to controls (age 1 year). Scale bar, 1 µm. ( G ) Overlapping NeuroD1 and NeuroD2 functions in postmigratory granule cell survival. (left) Normal cerebellar size and absence of granule cell apoptosis (TUNEL assay of GL in lobule 2) at P21 in conditional Neurod1 null mutants (NeuroD1 deficiency in postmigratory granule cells) with one wildtype copy of Neurod2 ( Neurod2 +/− ; Neurod1 fl/− ; Cre). (Middle) Neurod2 −/− mutants with one functional copy of Neurod1 ( Neurod2 −/− ; Neurod1 fl/− ) display reduced cerebellar size similar to Neurod2 −/− single mutants (see B ). Note that granule cell apoptosis has ceased by P21. (right) Reduced cerebellar size and granule cell apoptosis (arrowheads) in Neurod1/2 double mutants ( Neurod2 −/− ; Neurod1 fl/− ; Cre). Scale bars, 500 µm (H E stainings); 30 µm (TUNEL).
    Figure Legend Snippet: Surviving NeuroD2-deficient granule cells terminally differentiate and functionally integrate into excitatory cerebellar circuits. ( A ) Granule cells in Neurod2 −/− mutants extend claw-like telodendria (arrow) and axons projecting towards the ML (arrowhead). Golgi impregnation of sagittal sections (P25). Scale bar, 20 µm. ( B ) Fluorescent immunostaining for GABA Aα6 receptor on sagittal sections (lobule 5) from Neurod2 −/− mutants and controls (P25). (Right panels) magnification of boxed area in left panels. Scale bar, 600 µm. ( C ) Left: Schematic of PF EPSC recordings from Purkinje cells. PFs were stimulated in the ML (stim) while Purkinje cells were held in whole-cell voltage clamp (rec). Right: Representative traces of PF EPSCs from controls and Neurod2 −/− mutants. Paired-pulses reveal similar EPSC2 facilitation compared to EPSC1. ( D ) Left: Schematic of recordings of CF EPSCs from Purkinje cells. CFs were stimulated in the GL (stim) while Purkinje cells were held in whole-cell voltage clamp (rec). Right: Representative traces of CF EPSCs from control and Neurod2 −/− mutants. Paired-pulses reveal similar depression of EPSC2 compared to EPSC1. ( E ) Normal number and distribution of PFs and their terminals in Neurod2 −/− mutants compared to controls. Fluorescent immunostaining for Purkinje cell dendrites (PV) and PFs (calretinin, CR; or VGLUT1) (age 1 year). Note differences in PV staining due to loss of PV + MLIs in Neurod2 −/− mutants. Scale bars, 20 µm. (F) (Upper panels) Electron microscopy of granule cells in Neurod2 −/− mutants exhibits a similar distribution of nuclear heterochromatin compared to controls (age 1 year). Scale bar, 1.7 µm. (Lower panels) Ultrastructure of PF/Purkinje cell synapses (arrowheads) in Neurod2 −/− mutants compared to controls (age 1 year). Scale bar, 1 µm. ( G ) Overlapping NeuroD1 and NeuroD2 functions in postmigratory granule cell survival. (left) Normal cerebellar size and absence of granule cell apoptosis (TUNEL assay of GL in lobule 2) at P21 in conditional Neurod1 null mutants (NeuroD1 deficiency in postmigratory granule cells) with one wildtype copy of Neurod2 ( Neurod2 +/− ; Neurod1 fl/− ; Cre). (Middle) Neurod2 −/− mutants with one functional copy of Neurod1 ( Neurod2 −/− ; Neurod1 fl/− ) display reduced cerebellar size similar to Neurod2 −/− single mutants (see B ). Note that granule cell apoptosis has ceased by P21. (right) Reduced cerebellar size and granule cell apoptosis (arrowheads) in Neurod1/2 double mutants ( Neurod2 −/− ; Neurod1 fl/− ; Cre). Scale bars, 500 µm (H E stainings); 30 µm (TUNEL).

    Techniques Used: Immunostaining, Staining, Electron Microscopy, TUNEL Assay, Functional Assay

    15) Product Images from "Different TCR-induced T lymphocyte responses are potentiated by stiffness with variable sensitivity"

    Article Title: Different TCR-induced T lymphocyte responses are potentiated by stiffness with variable sensitivity

    Journal: eLife

    doi: 10.7554/eLife.23190

    Characterization of PA-gels and additionnal data on migration. ( A ) Measurement of the elastic modulus G´ of a PA-gel containing 5% acrylamide and 0.5% bis-acrylamide. The value associated to a given sample corresponds to the maximum of G´ as a function of the distance between the rheometer plates. The dotted line shows the mean value of the elastic shear modulus G´ = 2212 ± 79 Pa for n = 15 different samples of PA-gels at 5% acrylamide and 0.5% bis-acrylamide. ( B ) Coating of PA-gels and glass coverslips by biotinylated (b-fibronectin) or non biotinylated fibronectin quantified by immunofluorescence labeling. ( C ) Biotinylated antibody coating on streptavidin containing PA-gels of varying stiffness and neutravidin-coated glass coverslips quantified by immunofluorescence labeling (n samples : 14). ( D ) 5 min tracks of individual CD4 + T lymphoblasts on the 100 kPa gels coated with aCD3+aCD28+ICAM-1. Arrows indicate migrating cells and the arrowhead indicates an arrested cell. Scale bar: 10 μm. ( E ) Mean instantaneous velocities of migrating T cells on PA-gels of varying stiffness, coated with either ICAM-1 or aCD3+aCD28+ICAM-1, for a duration of 5 min (n cells : 50–100 for each condition from n Donors : 4). Boxes and whiskers for minimum and maximum are shown. For statistical analysis, unpaired parametric t-tests were performed: ****p-value
    Figure Legend Snippet: Characterization of PA-gels and additionnal data on migration. ( A ) Measurement of the elastic modulus G´ of a PA-gel containing 5% acrylamide and 0.5% bis-acrylamide. The value associated to a given sample corresponds to the maximum of G´ as a function of the distance between the rheometer plates. The dotted line shows the mean value of the elastic shear modulus G´ = 2212 ± 79 Pa for n = 15 different samples of PA-gels at 5% acrylamide and 0.5% bis-acrylamide. ( B ) Coating of PA-gels and glass coverslips by biotinylated (b-fibronectin) or non biotinylated fibronectin quantified by immunofluorescence labeling. ( C ) Biotinylated antibody coating on streptavidin containing PA-gels of varying stiffness and neutravidin-coated glass coverslips quantified by immunofluorescence labeling (n samples : 14). ( D ) 5 min tracks of individual CD4 + T lymphoblasts on the 100 kPa gels coated with aCD3+aCD28+ICAM-1. Arrows indicate migrating cells and the arrowhead indicates an arrested cell. Scale bar: 10 μm. ( E ) Mean instantaneous velocities of migrating T cells on PA-gels of varying stiffness, coated with either ICAM-1 or aCD3+aCD28+ICAM-1, for a duration of 5 min (n cells : 50–100 for each condition from n Donors : 4). Boxes and whiskers for minimum and maximum are shown. For statistical analysis, unpaired parametric t-tests were performed: ****p-value

    Techniques Used: Migration, Immunofluorescence, Labeling

    16) Product Images from "Generation and characterization of β1,2-gluco-oligosaccharide probes from Brucella abortus cyclic β-glucan and their recognition by C-type lectins of the immune system"

    Article Title: Generation and characterization of β1,2-gluco-oligosaccharide probes from Brucella abortus cyclic β-glucan and their recognition by C-type lectins of the immune system

    Journal: Glycobiology

    doi: 10.1093/glycob/cww041

    Carbohydrate microarray analysis of the interaction of C-type lectin receptors with CβG oligosaccharides. DC-SIGN-bio and DC-SIGNR-bio were tested at 50 µg mL −1 , serum MBP at 4 µg mL −1 and His-Dectin-1 at 20 µg/ml. The microarray consisted of lipid-linked gluco-oligosaccharide probes (AO-NGLs) printed in duplicate on nitrocellulose-coated glass slides. The linkage type and DP of the major component are indicated; their sequences are shown in Table I . The results are the means of fluorescence intensities of duplicate spots, printed at 2 and 5 fmol spot −1 (black and dark grey, respectively), and with the range indicated by error bars. This figure is available in black and white in print and in color at Glycobiology online.
    Figure Legend Snippet: Carbohydrate microarray analysis of the interaction of C-type lectin receptors with CβG oligosaccharides. DC-SIGN-bio and DC-SIGNR-bio were tested at 50 µg mL −1 , serum MBP at 4 µg mL −1 and His-Dectin-1 at 20 µg/ml. The microarray consisted of lipid-linked gluco-oligosaccharide probes (AO-NGLs) printed in duplicate on nitrocellulose-coated glass slides. The linkage type and DP of the major component are indicated; their sequences are shown in Table I . The results are the means of fluorescence intensities of duplicate spots, printed at 2 and 5 fmol spot −1 (black and dark grey, respectively), and with the range indicated by error bars. This figure is available in black and white in print and in color at Glycobiology online.

    Techniques Used: Microarray, Fluorescence

    17) Product Images from "Identification of Functional Cell Groups in the Abducens Nucleus of Monkey and Human by Perineuronal Nets and Choline Acetyltransferase Immunolabeling"

    Article Title: Identification of Functional Cell Groups in the Abducens Nucleus of Monkey and Human by Perineuronal Nets and Choline Acetyltransferase Immunolabeling

    Journal: Frontiers in Neuroanatomy

    doi: 10.3389/fnana.2018.00045

    Detailed views of corresponding planes of the right nVI in transverse monkey sections from different experiments. (A,B) Identification of INTs via retrograde labeling with choleratoxin subunit B (CTB) injection into the oculomotor nucleus (nIII; A,B , red) and their absence of ChAT-immunoreactivity ( B , green). Anterograde CTB-labeling from nIII highlights (A) the paramedian tractneurons (PMT) in the supragenual nucleus (SG) and (B) the “intrafascicular nucleus of the preabducens area” forming bridges between the medial nVI and midline ( A–F ; arrows). The PMT neurons are not ChAT-positive ( C , arrows), but are highlighted by acetylcholinesterase staining (AChE) in the SG and the “intrafascicular nucleus of the preabducens area” ( D , arrows). The appearance of PMT neurons is further demonstrated in corresponding sections stained for Nissl ( E , arrows) and CSPG ( F , arrows). Scale bar = 500 μm in ( E ; applies to A–F ). MLF, medial longitudinal fascicle; NVI, abducens nerve.
    Figure Legend Snippet: Detailed views of corresponding planes of the right nVI in transverse monkey sections from different experiments. (A,B) Identification of INTs via retrograde labeling with choleratoxin subunit B (CTB) injection into the oculomotor nucleus (nIII; A,B , red) and their absence of ChAT-immunoreactivity ( B , green). Anterograde CTB-labeling from nIII highlights (A) the paramedian tractneurons (PMT) in the supragenual nucleus (SG) and (B) the “intrafascicular nucleus of the preabducens area” forming bridges between the medial nVI and midline ( A–F ; arrows). The PMT neurons are not ChAT-positive ( C , arrows), but are highlighted by acetylcholinesterase staining (AChE) in the SG and the “intrafascicular nucleus of the preabducens area” ( D , arrows). The appearance of PMT neurons is further demonstrated in corresponding sections stained for Nissl ( E , arrows) and CSPG ( F , arrows). Scale bar = 500 μm in ( E ; applies to A–F ). MLF, medial longitudinal fascicle; NVI, abducens nerve.

    Techniques Used: Labeling, CtB Assay, Injection, Staining

    18) Product Images from "Cocaine withdrawal-induced trafficking of delta-opioid receptors in rat nucleus accumbens"

    Article Title: Cocaine withdrawal-induced trafficking of delta-opioid receptors in rat nucleus accumbens

    Journal:

    doi: 10.1016/j.brainres.2008.02.105

    Immunofluorescence microscopy showing localization of the D1R and DOR in the NAcbS
    Figure Legend Snippet: Immunofluorescence microscopy showing localization of the D1R and DOR in the NAcbS

    Techniques Used: Immunofluorescence, Microscopy

    Co-localization of the D1R and the DOR in the NAcbS and NAcbC
    Figure Legend Snippet: Co-localization of the D1R and the DOR in the NAcbS and NAcbC

    Techniques Used:

    19) Product Images from "Osteopontin protects against high phosphate-induced nephrocalcinosis and vascular calcification"

    Article Title: Osteopontin protects against high phosphate-induced nephrocalcinosis and vascular calcification

    Journal: Kidney international

    doi: 10.1016/j.kint.2015.12.046

    OPN expression is absent in the calcified vasculature of the KO+HP group OPN IHC of the abdominal aorta of the WT+HP mice (A) demonstrates no OPN expression in the vessel as expected in the absence of calcification. Despite the presence of mineral (arrow) the KO+HP aorta (B) is devoid of OPN. An adjacent section of the KO+HP aorta (C) is stained with Alizarin Red to highlight the mineral. Original magnification = 40X. Scale bar = 30 μm. L = lumen.
    Figure Legend Snippet: OPN expression is absent in the calcified vasculature of the KO+HP group OPN IHC of the abdominal aorta of the WT+HP mice (A) demonstrates no OPN expression in the vessel as expected in the absence of calcification. Despite the presence of mineral (arrow) the KO+HP aorta (B) is devoid of OPN. An adjacent section of the KO+HP aorta (C) is stained with Alizarin Red to highlight the mineral. Original magnification = 40X. Scale bar = 30 μm. L = lumen.

    Techniques Used: Expressing, Immunohistochemistry, Mouse Assay, Staining

    20) Product Images from "Teaching an old scaffold new tricks: Monobodies constructed using alternative surfaces of the FN3 scaffold"

    Article Title: Teaching an old scaffold new tricks: Monobodies constructed using alternative surfaces of the FN3 scaffold

    Journal: Journal of Molecular Biology

    doi: 10.1016/j.jmb.2011.12.019

    Oligomerization state and stability of monobodies. ( A ) Size-exclusion chromatograms of monobodies. The chromatographs are shown with vertical offsets for clarity. The labels show the identities of analyzed samples. The void volume (V 0 ) and elution positions
    Figure Legend Snippet: Oligomerization state and stability of monobodies. ( A ) Size-exclusion chromatograms of monobodies. The chromatographs are shown with vertical offsets for clarity. The labels show the identities of analyzed samples. The void volume (V 0 ) and elution positions

    Techniques Used:

    Monobody library designs and generated clones. Amino acid sequences of monobodies generated from the new “side and loop” library ( A ) and the “loop only” library ( B ). “X” denotes a mixture of 30% Tyr, 15%
    Figure Legend Snippet: Monobody library designs and generated clones. Amino acid sequences of monobodies generated from the new “side and loop” library ( A ) and the “loop only” library ( B ). “X” denotes a mixture of 30% Tyr, 15%

    Techniques Used: Generated, Clone Assay

    The crystal structures of monobodies originating from the two libraries. The structures are shown with the monobodies in similar orientations. ( A ) The structure of the SH13 monobody bound to the Abl SH2 domain depicted as in . ( B ) NMR-based epitope
    Figure Legend Snippet: The crystal structures of monobodies originating from the two libraries. The structures are shown with the monobodies in similar orientations. ( A ) The structure of the SH13 monobody bound to the Abl SH2 domain depicted as in . ( B ) NMR-based epitope

    Techniques Used: Nuclear Magnetic Resonance

    21) Product Images from "Vav-2 controls NFAT-dependent transcription inB- but not T-lymphocytes"

    Article Title: Vav-2 controls NFAT-dependent transcription inB- but not T-lymphocytes

    Journal: The EMBO Journal

    doi: 10.1093/emboj/19.22.6173

    Fig. 7. Ligation of CD19 results in tyrosine phosphorylation of Vav-2 and the activation of NFAT. ( A ) CD19 ligation induces tyrosine phosphorylation of Vav-2. Bal-17 B cells were incubated with the indicated doses of biotinylated Fab fragment of antibody to CD19 and then stimulated by the addition of soluble avidin for 2 min. Cells were lysed and immunoprecipitates prepared with non-immune (NI) or polyclonal Vav-2 antibodies, and immunoblotted with antibody to phosphotyrosine (top panel). The blot was stripped and reprobed with pooled mAbs to Vav-2. ( B ) Tyrosine phosphorylation of Vav-2 is enhanced by co-ligation of CD19 to BCR. Bal-17 B cells were incubated with the indicated doses of either control Fab, biotinylated Fab anti-CD19, or biotinylated Fab anti-κ alone or combined with 0.2 µg of biotinylated Fab anti-CD19, followed by crosslinking with soluble avidin for 2 min. Cells were lysed and immunoprecipitates prepared with non-immune (NI) or polyclonal Vav-2 and immuno blotted with antibody to phosphotyrosine (top panel). The blot was stripped and reprobed as above. ( C ) This experiment was performed as in (B) except that the stimulating dose of Fab anti-CD19 was 5 µg/ml. ( D ) Vav-2 potentiates CD19 induction of NFAT. Bal-17 B cells were co-transfected with p(NFAT) 3 IL-2-luc together with 1 µg of empty vector or plasmids encoding Vav-2, L212R Vav-2 or R688A Vav-2. Cells were cultured in medium alone (unstimulated) or were incubated with 1D3 anti-CD19, which was crosslinked with F(ab′) 2 goat anti-rat IgG before cell lysates were prepared and assayed for luciferase activity.
    Figure Legend Snippet: Fig. 7. Ligation of CD19 results in tyrosine phosphorylation of Vav-2 and the activation of NFAT. ( A ) CD19 ligation induces tyrosine phosphorylation of Vav-2. Bal-17 B cells were incubated with the indicated doses of biotinylated Fab fragment of antibody to CD19 and then stimulated by the addition of soluble avidin for 2 min. Cells were lysed and immunoprecipitates prepared with non-immune (NI) or polyclonal Vav-2 antibodies, and immunoblotted with antibody to phosphotyrosine (top panel). The blot was stripped and reprobed with pooled mAbs to Vav-2. ( B ) Tyrosine phosphorylation of Vav-2 is enhanced by co-ligation of CD19 to BCR. Bal-17 B cells were incubated with the indicated doses of either control Fab, biotinylated Fab anti-CD19, or biotinylated Fab anti-κ alone or combined with 0.2 µg of biotinylated Fab anti-CD19, followed by crosslinking with soluble avidin for 2 min. Cells were lysed and immunoprecipitates prepared with non-immune (NI) or polyclonal Vav-2 and immuno blotted with antibody to phosphotyrosine (top panel). The blot was stripped and reprobed as above. ( C ) This experiment was performed as in (B) except that the stimulating dose of Fab anti-CD19 was 5 µg/ml. ( D ) Vav-2 potentiates CD19 induction of NFAT. Bal-17 B cells were co-transfected with p(NFAT) 3 IL-2-luc together with 1 µg of empty vector or plasmids encoding Vav-2, L212R Vav-2 or R688A Vav-2. Cells were cultured in medium alone (unstimulated) or were incubated with 1D3 anti-CD19, which was crosslinked with F(ab′) 2 goat anti-rat IgG before cell lysates were prepared and assayed for luciferase activity.

    Techniques Used: Ligation, Activation Assay, Incubation, Avidin-Biotin Assay, Transfection, Plasmid Preparation, Cell Culture, Luciferase, Activity Assay

    Fig. 8. Vav-2 interacts with tyrosine phosphorylated CD19. ( A ) or antibody to Vav-2 (αVav-2) were incubated with lysates prepared from COS7 cells that had been transfected with Vav-2. The resulting complexes were probed with pooled mAbs to Vav-2. A portion of the transfected COS7 cell lysate (WCL) was loaded onto the gel as a control. ( B ) Y*391 binds directly to the SH2 domain of Vav-2. Equal amounts of fusion proteins representing GST alone or the SH2 domain of either Vav-1 or Vav-2 immobilized on nitrocellulose were incubated with biotinylated peptide Y*391 (top panel), biotinylated peptide Y391 (middle panel) or anti-GST (bottom panel). ( C ) Y*391 specifically inhibits co-precipitation of a 100–110 kDa tyrosine phosphoprotein with Vav-2. Bal-17 B cells were incubated with either 10 µg of control Fab (–) or 5 µg of biotinylated Fab anti-κ combined with 5 µg of biotinylated Fab anti-CD19 (+), which were crosslinked by the addition of soluble avidin for 2 min. Following cell lysis, immunoprecipitates were prepared with non-immune or polyclonal Vav-2 antibodies in the absence of peptide or in the presence of 100 µM Y391 or Y*391 and immunoblotted with antibody to phosphotyrosine (top panel). The positions of the co-precipitating 100–110 kDa proteins are marked with curly brackets. The blot was stripped and reprobed with pooled mAbs to Vav-2 (bottom panel).
    Figure Legend Snippet: Fig. 8. Vav-2 interacts with tyrosine phosphorylated CD19. ( A ) or antibody to Vav-2 (αVav-2) were incubated with lysates prepared from COS7 cells that had been transfected with Vav-2. The resulting complexes were probed with pooled mAbs to Vav-2. A portion of the transfected COS7 cell lysate (WCL) was loaded onto the gel as a control. ( B ) Y*391 binds directly to the SH2 domain of Vav-2. Equal amounts of fusion proteins representing GST alone or the SH2 domain of either Vav-1 or Vav-2 immobilized on nitrocellulose were incubated with biotinylated peptide Y*391 (top panel), biotinylated peptide Y391 (middle panel) or anti-GST (bottom panel). ( C ) Y*391 specifically inhibits co-precipitation of a 100–110 kDa tyrosine phosphoprotein with Vav-2. Bal-17 B cells were incubated with either 10 µg of control Fab (–) or 5 µg of biotinylated Fab anti-κ combined with 5 µg of biotinylated Fab anti-CD19 (+), which were crosslinked by the addition of soluble avidin for 2 min. Following cell lysis, immunoprecipitates were prepared with non-immune or polyclonal Vav-2 antibodies in the absence of peptide or in the presence of 100 µM Y391 or Y*391 and immunoblotted with antibody to phosphotyrosine (top panel). The positions of the co-precipitating 100–110 kDa proteins are marked with curly brackets. The blot was stripped and reprobed with pooled mAbs to Vav-2 (bottom panel).

    Techniques Used: Incubation, Transfection, Avidin-Biotin Assay, Lysis

    Fig. 1. Vav-2 is tyrosine phosphorylated after antigen–receptor ligation in primary B lymphocytes. ( A ) Vav-2 displays similar kinetics of tyrosine phosphorylation to Vav-1 in response to BCR ligation. Resting B cells were purified from mouse spleen and incubated with 10 µg/ml polyclonal F(ab′) 2 anti-IgM and aliquots of cells were removed at timed intervals to make lysates. Lysates were divided in half and immunoprecipitates were prepared with irrelevant non-immune (NI) and polyclonal Vav-1- or Vav-2-specific antibodies. These were immunoblotted with antibody to phosphotyrosine (top panels). The blots were stripped and reprobed with monoclonal anti-Vav-1 or pooled mAbs specific for Vav-2 (bottom panels). ( B ) Vav-2 is tyrosine phosphorylated in a dose-dependent manner. B cells were isolated from mouse spleen and incubated with incremental doses of polyclonal F(ab′) 2 anti-IgM. After 2 min, cells were lysed and divided in half; immunoprecipitates were prepared with non-immune (NI) and Vav-1- or Vav-2-specific polyclonal antibodies, and immunoblotted with antibody to phosphotyrosine (top panels). The blots were stripped and reprobed as above (bottom panels).
    Figure Legend Snippet: Fig. 1. Vav-2 is tyrosine phosphorylated after antigen–receptor ligation in primary B lymphocytes. ( A ) Vav-2 displays similar kinetics of tyrosine phosphorylation to Vav-1 in response to BCR ligation. Resting B cells were purified from mouse spleen and incubated with 10 µg/ml polyclonal F(ab′) 2 anti-IgM and aliquots of cells were removed at timed intervals to make lysates. Lysates were divided in half and immunoprecipitates were prepared with irrelevant non-immune (NI) and polyclonal Vav-1- or Vav-2-specific antibodies. These were immunoblotted with antibody to phosphotyrosine (top panels). The blots were stripped and reprobed with monoclonal anti-Vav-1 or pooled mAbs specific for Vav-2 (bottom panels). ( B ) Vav-2 is tyrosine phosphorylated in a dose-dependent manner. B cells were isolated from mouse spleen and incubated with incremental doses of polyclonal F(ab′) 2 anti-IgM. After 2 min, cells were lysed and divided in half; immunoprecipitates were prepared with non-immune (NI) and Vav-1- or Vav-2-specific polyclonal antibodies, and immunoblotted with antibody to phosphotyrosine (top panels). The blots were stripped and reprobed as above (bottom panels).

    Techniques Used: Ligation, Purification, Incubation, Isolation

    22) Product Images from "Critical Role for Glial Cells in the Propagation and Spread of Lymphocytic Choriomeningitis Virus in the Developing Rat Brain"

    Article Title: Critical Role for Glial Cells in the Propagation and Spread of Lymphocytic Choriomeningitis Virus in the Developing Rat Brain

    Journal: Journal of Virology

    doi: 10.1128/JVI.76.13.6618-6635.2002

    Fifty-micrometer-thick sections through the hippocampal formation immunohistochemically stained for LCMV (panels A, B, D, E, F, and G) or GFAP (panel C). LCMV infection in the hippocampus initially involves astrocytes. The granule cells of the dentate gyrus become chronically infected. (A) Low-power image of hippocampal region at PD8 shows infection within the ependyma, parenchymal cells adjacent to the ependyma (double arrowheads), and fornix (arrowhead). The dentate gyrus (arrow) is not labeled. (B) Area beneath the ependyma, represented by the box in panel A, shows that the infected cells have the morphology of astrocytes (arrowheads). (C) A section adjacent to panel B immunohistochemically labeled for GFAP, a marker for astrocytes. The GFAP-positive cells in panel C have the same morphology as the LCMV-infected cells in panel B, indicating that the infected parenchymal cells on PD8 are astrocytes. (D) By PD18, infection in the hippocampal region is less intense but still present beneath the ependyma (double arrowheads) and in the fornix (arrowhead). The dentate gyrus stratum granulosum (arrow) now shows evidence of infection. (E) Higher magnification of box in panel D shows that granule cells of the dentate gyrus (arrows), and nearby astrocytes (arrowheads) are both infected with LCMV on PD18. (F) By PD49, infection has been cleared from the hippocampal region, including the fornix (arrowhead), except for granule cells of the dentate gyrus (arrow), where viral antigen persists. (G) Higher magnification of the box in panel F demonstrates that cell bodies and dendrites of dentate granule cells (arrows) and a few hilar neurons (double arrowheads) are still infected. The virus has been cleared from glial cells. Bars, 1 mm (A, D, and F); 20 μm (B and C); 100 μm (E and G).
    Figure Legend Snippet: Fifty-micrometer-thick sections through the hippocampal formation immunohistochemically stained for LCMV (panels A, B, D, E, F, and G) or GFAP (panel C). LCMV infection in the hippocampus initially involves astrocytes. The granule cells of the dentate gyrus become chronically infected. (A) Low-power image of hippocampal region at PD8 shows infection within the ependyma, parenchymal cells adjacent to the ependyma (double arrowheads), and fornix (arrowhead). The dentate gyrus (arrow) is not labeled. (B) Area beneath the ependyma, represented by the box in panel A, shows that the infected cells have the morphology of astrocytes (arrowheads). (C) A section adjacent to panel B immunohistochemically labeled for GFAP, a marker for astrocytes. The GFAP-positive cells in panel C have the same morphology as the LCMV-infected cells in panel B, indicating that the infected parenchymal cells on PD8 are astrocytes. (D) By PD18, infection in the hippocampal region is less intense but still present beneath the ependyma (double arrowheads) and in the fornix (arrowhead). The dentate gyrus stratum granulosum (arrow) now shows evidence of infection. (E) Higher magnification of box in panel D shows that granule cells of the dentate gyrus (arrows), and nearby astrocytes (arrowheads) are both infected with LCMV on PD18. (F) By PD49, infection has been cleared from the hippocampal region, including the fornix (arrowhead), except for granule cells of the dentate gyrus (arrow), where viral antigen persists. (G) Higher magnification of the box in panel F demonstrates that cell bodies and dendrites of dentate granule cells (arrows) and a few hilar neurons (double arrowheads) are still infected. The virus has been cleared from glial cells. Bars, 1 mm (A, D, and F); 20 μm (B and C); 100 μm (E and G).

    Techniques Used: Staining, Infection, Labeling, Marker

    23) Product Images from "Input-Specific Immunolocalization of Differentially Phosphorylated Kv4.2 in the Mouse Brain"

    Article Title: Input-Specific Immunolocalization of Differentially Phosphorylated Kv4.2 in the Mouse Brain

    Journal: Learning & Memory

    doi:

    Mouse brain coronal sections. Staining for ERK triply phosphorylated Kv4.2 ( A ), carboxy-terminal PKA-phosphorylated Kv4.2 ( B ) and amino-terminal PKA-phosphorylated Kv4.2 ( C ). The right half in each brain displays immunoreactivity to the appropriate antibody, whereas the left half is representative of the immunoreactivity seen when the antibody is preincubated with its appropriate antigen. In general, staining across antibodies is strong in areas consistent with those described by the pattern of total Kv4.2 staining. Strong immunoreactivity is seen in the hippocampus, thalamus, medial habenular nucleus, striatum, amygdala, cortex, and cerebellum.
    Figure Legend Snippet: Mouse brain coronal sections. Staining for ERK triply phosphorylated Kv4.2 ( A ), carboxy-terminal PKA-phosphorylated Kv4.2 ( B ) and amino-terminal PKA-phosphorylated Kv4.2 ( C ). The right half in each brain displays immunoreactivity to the appropriate antibody, whereas the left half is representative of the immunoreactivity seen when the antibody is preincubated with its appropriate antigen. In general, staining across antibodies is strong in areas consistent with those described by the pattern of total Kv4.2 staining. Strong immunoreactivity is seen in the hippocampus, thalamus, medial habenular nucleus, striatum, amygdala, cortex, and cerebellum.

    Techniques Used: Staining

    Cortex and Cerebellum. Staining of ERK triply phosphorylated Kv4.2 ( A,D ), carboxy-terminal PKA-phosphorylated Kv4.2 ( B,E ), and amino-terminal PKA-phosphorylated Kv4.2 ( C,F ) in the cortex ( A,B,C ) and cerebellum ( D,E,F ) at 100×. In the cortex, ERK triply phosphorylated Kv4.2 antibodies show predominant staining in layer IV of the cortex with possible evidence of barrels ( A ). Carboxy-terminal PKA-phosphorylated Kv4.2 staining shows weak immunoreactivity in layers V and VI ( B ). Amino-terminal PKA-phosphorylated Kv4.2 staining displays immunoreactivity in layers IV, V, and VI with the strongest signal in layers V and VI ( C ). In the cerebellum, staining with the ERK triply phosphorylated and carboxy-terminal PKA-phosphorylated Kv4.2 is nearly identical. Strong staining can be visualized in the granular layer, however, staining is absent from the Purkinje cell bodies and is only minor in the molecular layer ( D,E ). In contrast to the other two antibodies, amino-terminal PKA-phosphorylated Kv4.2 antibodies recognize the molecular layer, and staining is highly reduced in the granular layer. Purkinje cell bodies are void of immunoreactivity.
    Figure Legend Snippet: Cortex and Cerebellum. Staining of ERK triply phosphorylated Kv4.2 ( A,D ), carboxy-terminal PKA-phosphorylated Kv4.2 ( B,E ), and amino-terminal PKA-phosphorylated Kv4.2 ( C,F ) in the cortex ( A,B,C ) and cerebellum ( D,E,F ) at 100×. In the cortex, ERK triply phosphorylated Kv4.2 antibodies show predominant staining in layer IV of the cortex with possible evidence of barrels ( A ). Carboxy-terminal PKA-phosphorylated Kv4.2 staining shows weak immunoreactivity in layers V and VI ( B ). Amino-terminal PKA-phosphorylated Kv4.2 staining displays immunoreactivity in layers IV, V, and VI with the strongest signal in layers V and VI ( C ). In the cerebellum, staining with the ERK triply phosphorylated and carboxy-terminal PKA-phosphorylated Kv4.2 is nearly identical. Strong staining can be visualized in the granular layer, however, staining is absent from the Purkinje cell bodies and is only minor in the molecular layer ( D,E ). In contrast to the other two antibodies, amino-terminal PKA-phosphorylated Kv4.2 antibodies recognize the molecular layer, and staining is highly reduced in the granular layer. Purkinje cell bodies are void of immunoreactivity.

    Techniques Used: Staining

    Antibody specificity. Western blotting was performed on hippocampal homogenate membrane preps by use of antibodies against ERK triply phosphorylated Kv4.2 ( A ), carboxy-terminal PKA-phosphorylated Kv4.2 ( B ), and amino-terminanl PKA-phosphorylated Kv4.2 ( C ). Preincubation with the corresponding antigen blocked 90%–100% of immunoreactivity. In C , the hippocampus was first treated with forskolin to improve the immunoreactivity.
    Figure Legend Snippet: Antibody specificity. Western blotting was performed on hippocampal homogenate membrane preps by use of antibodies against ERK triply phosphorylated Kv4.2 ( A ), carboxy-terminal PKA-phosphorylated Kv4.2 ( B ), and amino-terminanl PKA-phosphorylated Kv4.2 ( C ). Preincubation with the corresponding antigen blocked 90%–100% of immunoreactivity. In C , the hippocampus was first treated with forskolin to improve the immunoreactivity.

    Techniques Used: Western Blot

    Higher magnification of area CA3. Staining of ERK triply phosphorylated Kv4.2 at 200× ( A ) and 100× ( D ), carboxy-terminal PKA-phosphorylated Kv4.2 at 200× ( B ) and 100× ( E ), and amino-terminal PKA-phosphorylated Kv4.2 at 200× ( C ). Heavy staining of CA3 neuronal soma can be seen in A , with a dearth of immunoreactivity in this same area in B and C. D and E show the contrast in immunoreactivity in the stratum lucidum between the ERK triply phosphorylated Kv4.2 antibody ( D ) and the carboxy-terminal PKA-phosphorylated Kv4.2 antibody ( E ).
    Figure Legend Snippet: Higher magnification of area CA3. Staining of ERK triply phosphorylated Kv4.2 at 200× ( A ) and 100× ( D ), carboxy-terminal PKA-phosphorylated Kv4.2 at 200× ( B ) and 100× ( E ), and amino-terminal PKA-phosphorylated Kv4.2 at 200× ( C ). Heavy staining of CA3 neuronal soma can be seen in A , with a dearth of immunoreactivity in this same area in B and C. D and E show the contrast in immunoreactivity in the stratum lucidum between the ERK triply phosphorylated Kv4.2 antibody ( D ) and the carboxy-terminal PKA-phosphorylated Kv4.2 antibody ( E ).

    Techniques Used: Staining

    Higher magnification of area CA1. Staining of ERK triply phosphorylated Kv4.2 at 100× ( A ), carboxy-terminal PKA-phosphorylated Kv4.2 at 100× ( B ), and amino-terminal PKA-phosphorylated Kv4.2 at 100× ( C ). Strong immunoreactivity in the CA1 stratum oriens (so) and stratum radiatum (sr) can be seen in A and B with a dearth of immunoreactivity in these same layers in C . However, there is a relative dearth of immunoreactivity in the stratum lacunosum moleculare (slm) in A and B , whereas immunoreactivity is strong in the slm in C . The immunoreactivity in the molecular layer of the dentate gyrus (ml-dg) is increased in B as compared with A and C . (sp) Stratum pyramidali.
    Figure Legend Snippet: Higher magnification of area CA1. Staining of ERK triply phosphorylated Kv4.2 at 100× ( A ), carboxy-terminal PKA-phosphorylated Kv4.2 at 100× ( B ), and amino-terminal PKA-phosphorylated Kv4.2 at 100× ( C ). Strong immunoreactivity in the CA1 stratum oriens (so) and stratum radiatum (sr) can be seen in A and B with a dearth of immunoreactivity in these same layers in C . However, there is a relative dearth of immunoreactivity in the stratum lacunosum moleculare (slm) in A and B , whereas immunoreactivity is strong in the slm in C . The immunoreactivity in the molecular layer of the dentate gyrus (ml-dg) is increased in B as compared with A and C . (sp) Stratum pyramidali.

    Techniques Used: Staining

    Kv4.2 structure and peptide antigens. Schematic structure of Kv4.2 with highlighted ERK and PKA phosphorylation sites. The ERK triply phosphorylated antibody was made against a peptide running from AA586 to AA618, which includes the ERK phosphorylation sites Thr 602 , Thr 607 , and Ser 616 . The carboxy-terminal PKA-phosphorylated Kv4.2 antibody was made against a peptide running from AA546 to AA548, which includes a PKA phosphorylation site at Ser 552 . The amino-terminal PKA-phosphorylated Kv4.2 antibody was made against a peptide running from AA32 to AA44, which includes a PKA phosphorylation site at Thr 38 .
    Figure Legend Snippet: Kv4.2 structure and peptide antigens. Schematic structure of Kv4.2 with highlighted ERK and PKA phosphorylation sites. The ERK triply phosphorylated antibody was made against a peptide running from AA586 to AA618, which includes the ERK phosphorylation sites Thr 602 , Thr 607 , and Ser 616 . The carboxy-terminal PKA-phosphorylated Kv4.2 antibody was made against a peptide running from AA546 to AA548, which includes a PKA phosphorylation site at Ser 552 . The amino-terminal PKA-phosphorylated Kv4.2 antibody was made against a peptide running from AA32 to AA44, which includes a PKA phosphorylation site at Thr 38 .

    Techniques Used:

    24) Product Images from "Morbillivirus Infection of the Mouse Central Nervous System Induces Region-Specific Upregulation of MMPs and TIMPs Correlated to Inflammatory Cytokine Expression"

    Article Title: Morbillivirus Infection of the Mouse Central Nervous System Induces Region-Specific Upregulation of MMPs and TIMPs Correlated to Inflammatory Cytokine Expression

    Journal: Journal of Virology

    doi: 10.1128/JVI.75.17.8268-8282.2001

    Expression of gelatinases MMP-2 and MMP-9 in microdissected brain structures. (a) Gelatin zymography. To increase their concentration, the gelatinases were purified from brain structure lysates on gelatin-Sepharose beads, using the method of Zhang and Gottschall (67), and loaded on a 9% polyacrylamide gel containing gelatin (0.07%) and activated (20 h at 37°C), and then the gels were stained. Constitutive expression of active MMP-2 (65 kDa) was detected in all sham-inoculated structures, while faint MMP-9 proteolytic activity (92 kDa) was seen only in the mesencephalon, brain stem, cerebellum, and spinal cord. MMP-2 and pro-MMP-9 were markedly upregulated in infected brain structures, in particular in the rostral part of the brain. (b) Densitometric analysis. Data expressed as the ratio of infected/sham-inoculated normalized values (relative units) showed upregulation of MMP-2 and MMP-9 mainly in the hippocampus and, to a lesser extent, in the cortex and hypothalamus of infected mice. cx, cortex; mes, mesencephalon; hip, hippocampus; hyp, hypothalamus; bs, brain stem; cb, cerebellum; sc, spinal cord. (c) APMA treatment of hippocampal lysates. To determine if gelatinases are expressed as prozymogens or active enzymes, hippocampal lysates from sham-inoculated and infected mice at 14 dpi were treated with APMA and then electrophoresed as above. The zymograms for the untreated samples (lanes 1 and 2) showed two clear bands of respective apparent molecular masses of 65 and 92 kDa, presumably corresponding to active MMP-2 and pro-MMP-9, which were strongly upregulated in infected hippocampus (lane 2). In the APMA-treated samples (lanes 3 and 4), the 92-kDa product disappeared while a 75-kDa apparent molecular mass product became visible; the 65-kDa product corresponds to active MMP-2 that remained unchanged. Culture supernatant from PMA-treated BHK21 cells (containing the MMP-2 active forms) and TNF-α-treated DEV cells (containing the MMP-9 active form) were used as positive controls. Lanes 1 and 3, hippocampus from a sham-inoculated mouse; lanes 2 and 4, hippocampus from an infected mouse
    Figure Legend Snippet: Expression of gelatinases MMP-2 and MMP-9 in microdissected brain structures. (a) Gelatin zymography. To increase their concentration, the gelatinases were purified from brain structure lysates on gelatin-Sepharose beads, using the method of Zhang and Gottschall (67), and loaded on a 9% polyacrylamide gel containing gelatin (0.07%) and activated (20 h at 37°C), and then the gels were stained. Constitutive expression of active MMP-2 (65 kDa) was detected in all sham-inoculated structures, while faint MMP-9 proteolytic activity (92 kDa) was seen only in the mesencephalon, brain stem, cerebellum, and spinal cord. MMP-2 and pro-MMP-9 were markedly upregulated in infected brain structures, in particular in the rostral part of the brain. (b) Densitometric analysis. Data expressed as the ratio of infected/sham-inoculated normalized values (relative units) showed upregulation of MMP-2 and MMP-9 mainly in the hippocampus and, to a lesser extent, in the cortex and hypothalamus of infected mice. cx, cortex; mes, mesencephalon; hip, hippocampus; hyp, hypothalamus; bs, brain stem; cb, cerebellum; sc, spinal cord. (c) APMA treatment of hippocampal lysates. To determine if gelatinases are expressed as prozymogens or active enzymes, hippocampal lysates from sham-inoculated and infected mice at 14 dpi were treated with APMA and then electrophoresed as above. The zymograms for the untreated samples (lanes 1 and 2) showed two clear bands of respective apparent molecular masses of 65 and 92 kDa, presumably corresponding to active MMP-2 and pro-MMP-9, which were strongly upregulated in infected hippocampus (lane 2). In the APMA-treated samples (lanes 3 and 4), the 92-kDa product disappeared while a 75-kDa apparent molecular mass product became visible; the 65-kDa product corresponds to active MMP-2 that remained unchanged. Culture supernatant from PMA-treated BHK21 cells (containing the MMP-2 active forms) and TNF-α-treated DEV cells (containing the MMP-9 active form) were used as positive controls. Lanes 1 and 3, hippocampus from a sham-inoculated mouse; lanes 2 and 4, hippocampus from an infected mouse

    Techniques Used: Expressing, Zymography, Concentration Assay, Purification, Staining, Activity Assay, Infection, Mouse Assay

    Immunodetection of gelatinases MMP-2 and MMP-9 and localization of gelatinolytic activity by ISZ. Immunodetection (IHC) of MMP-9 and MMP-2 using antibodies against the whole protein (reactive with both the proenzyme and enzyme forms) showed MMP-9 to be present in the hippocampus (a) at the level of the CA3 pyramidal layer. At the cellular level, MMP-9 was mainly found in neurons, identified by their size and localization (panel a and insert), as confirmed using double labeling. (c) MMP-9 (green), indicated by white arrows; (d) neuronal marker MAP-2 (red), indicated by white arrows. It is noteworthy that all the MAP-2-positive cells are not always MMP-9 positive. MMP-2 was mainly located in astrocyte-type cells (panel b and insert). For ISZ, the quenched fluorescent substrate (gelatin) was added directly to tissue sections, and enzymatic activity was detected by the unmasked fluorescence. Cellular gelatinolytic activity was faint and diffuse in brain sections from sham-inoculated mice (e) and was markedly enhanced in the cells of the pyramidal layers of the hippocampus from CDV-infected mice (f), which is also shown at a higher magnification (insert in panel f versus that in panel e). Magnifications, ×28 (a, b, e, f); ×40 (c, d, and inserts in panels a, b, e, and f).
    Figure Legend Snippet: Immunodetection of gelatinases MMP-2 and MMP-9 and localization of gelatinolytic activity by ISZ. Immunodetection (IHC) of MMP-9 and MMP-2 using antibodies against the whole protein (reactive with both the proenzyme and enzyme forms) showed MMP-9 to be present in the hippocampus (a) at the level of the CA3 pyramidal layer. At the cellular level, MMP-9 was mainly found in neurons, identified by their size and localization (panel a and insert), as confirmed using double labeling. (c) MMP-9 (green), indicated by white arrows; (d) neuronal marker MAP-2 (red), indicated by white arrows. It is noteworthy that all the MAP-2-positive cells are not always MMP-9 positive. MMP-2 was mainly located in astrocyte-type cells (panel b and insert). For ISZ, the quenched fluorescent substrate (gelatin) was added directly to tissue sections, and enzymatic activity was detected by the unmasked fluorescence. Cellular gelatinolytic activity was faint and diffuse in brain sections from sham-inoculated mice (e) and was markedly enhanced in the cells of the pyramidal layers of the hippocampus from CDV-infected mice (f), which is also shown at a higher magnification (insert in panel f versus that in panel e). Magnifications, ×28 (a, b, e, f); ×40 (c, d, and inserts in panels a, b, e, and f).

    Techniques Used: Immunodetection, Activity Assay, Immunohistochemistry, Labeling, Marker, Fluorescence, Mouse Assay, Infection

    MMP-2, MMP-9, and MT1-MMP mRNA expression analyzed by RT-PCR and densitometry. Total RNAs (0.5 μg) extracted from microdissected brain structures from infected and sham-inoculated mice were subjected to RT-PCR. After electrophoresis on an agarose gel and electrotransfer, Southern blotting of the amplicons was performed. Hybridization of specific internal radiolabeled probes allowed the semiquantification of each PCR product. MMP expression was then analyzed by phosphorimaging densitometry. (a) The relative mRNA content for each amplicon was calculated as a fraction of the levels of the housekeeping gene G3PDH mRNA (normalized values), and the results were expressed as a ratio of levels in infected mice relative to those in sham-inoculated mice (relative units). Only slight MMP-2 and MMP-9 upregulation was seen in brain structures of CDV-infected mice, the difference not being significant in the Mann-Whitney test (b). In contrast, marked upregulation of MT1-MMP was seen in infected mice, mainly in the rostral brain (cortex, hippocampus, and hypothalamus), the difference being statistically significant in the cortex and hypothalamus ( P
    Figure Legend Snippet: MMP-2, MMP-9, and MT1-MMP mRNA expression analyzed by RT-PCR and densitometry. Total RNAs (0.5 μg) extracted from microdissected brain structures from infected and sham-inoculated mice were subjected to RT-PCR. After electrophoresis on an agarose gel and electrotransfer, Southern blotting of the amplicons was performed. Hybridization of specific internal radiolabeled probes allowed the semiquantification of each PCR product. MMP expression was then analyzed by phosphorimaging densitometry. (a) The relative mRNA content for each amplicon was calculated as a fraction of the levels of the housekeeping gene G3PDH mRNA (normalized values), and the results were expressed as a ratio of levels in infected mice relative to those in sham-inoculated mice (relative units). Only slight MMP-2 and MMP-9 upregulation was seen in brain structures of CDV-infected mice, the difference not being significant in the Mann-Whitney test (b). In contrast, marked upregulation of MT1-MMP was seen in infected mice, mainly in the rostral brain (cortex, hippocampus, and hypothalamus), the difference being statistically significant in the cortex and hypothalamus ( P

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Infection, Mouse Assay, Electrophoresis, Agarose Gel Electrophoresis, Electrotransfer, Southern Blot, Hybridization, Polymerase Chain Reaction, Amplification, MANN-WHITNEY

    25) Product Images from "Age-Dependent Anti-migraine Effects of Valproic Acid and Topiramate in Rats"

    Article Title: Age-Dependent Anti-migraine Effects of Valproic Acid and Topiramate in Rats

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2018.01095

    c-Fos-ir in trigeminocervical complex (TCC) and CGRP-ir in trigeminal ganglia (TG) and dura of adult and pediatric rats treated with topiramate (TPM). (A) The immunochemical stain of c-Fos shows c-Fos-ir at various dosages of TPM in both age groups. (B) Intracisternal capsaicin ( i.c. ) significantly induced c-Fos-ir in TCC of the adult compared to sham (AC vs. AS, p = 0.021). The capsaicin induced c-Fos-ir is significantly suppressed by TPM in adult at either 30 (ACT30 vs. AC, p = 0.043) or 100 (ACT100 vs. AC, p = 0.021) mg/kg, but not 10 mg/kg (ACT10 vs. AC). (C) Capsaicin significantly induced c-Fos-ir in TCC of the pediatric (PC vs. PS, p = 0.021). The capsaicin induced c-Fos-ir is significantly suppressed by TPM in pediatric at either 30 (PCT30 vs. AC, p = 0.021) or 100 (ACT100 vs. AC, p = 0.021) mg/kg, but not 10 mg/kg (PCT10 vs. AC). (D) The immunochemical stain of TG shows CGRP-ir at various dosages of TPM in both age groups. (E) Capsaicin significantly induced CGRP-ir in TG of the adult (AC vs. AS, p = 0.046). The capsaicin induced CGRP-ir of TG is significantly suppressed by TPM in the adult group at all the three doses (ACT vs. AC, p
    Figure Legend Snippet: c-Fos-ir in trigeminocervical complex (TCC) and CGRP-ir in trigeminal ganglia (TG) and dura of adult and pediatric rats treated with topiramate (TPM). (A) The immunochemical stain of c-Fos shows c-Fos-ir at various dosages of TPM in both age groups. (B) Intracisternal capsaicin ( i.c. ) significantly induced c-Fos-ir in TCC of the adult compared to sham (AC vs. AS, p = 0.021). The capsaicin induced c-Fos-ir is significantly suppressed by TPM in adult at either 30 (ACT30 vs. AC, p = 0.043) or 100 (ACT100 vs. AC, p = 0.021) mg/kg, but not 10 mg/kg (ACT10 vs. AC). (C) Capsaicin significantly induced c-Fos-ir in TCC of the pediatric (PC vs. PS, p = 0.021). The capsaicin induced c-Fos-ir is significantly suppressed by TPM in pediatric at either 30 (PCT30 vs. AC, p = 0.021) or 100 (ACT100 vs. AC, p = 0.021) mg/kg, but not 10 mg/kg (PCT10 vs. AC). (D) The immunochemical stain of TG shows CGRP-ir at various dosages of TPM in both age groups. (E) Capsaicin significantly induced CGRP-ir in TG of the adult (AC vs. AS, p = 0.046). The capsaicin induced CGRP-ir of TG is significantly suppressed by TPM in the adult group at all the three doses (ACT vs. AC, p

    Techniques Used: Staining, Activated Clotting Time Assay

    c-Fos-ir in trigeminocervical complex (TCC) and CGRP-ir in trigeminal ganglia (TG) and dura of adult and pediatric rats treated with valproic acid (VPA). (A) The immunochemical stain of c-Fos shows c-Fos-ir at various dosages in both age groups. (B) Intracisternal capsaicin ( i.c. ) significantly induced c-Fos-ir in TCC of the adult compared to sham (AC vs. AS, p = 0.021). The capsaicin induced c-Fos-ir is not significantly suppressed by VPA in adult at either 30 (ACV30 vs. AC) or 100 (ACV100 vs. AC) mg/kg. (C) Capsaicin i.c. significantly induced c-Fos-ir in TCC of the pediatric (PC vs. PS, p = 0.021). The capsaicin induced c-Fos-ir is significantly suppressed by VPA in pediatric at 100 mg/kg (PCV100 vs. PC, p = 0.021) but not 30 mg/kg (PCV30 vs. PC). (D) The immunochemical stain of TG shows CGRP-ir at various dosages in both age groups. (E) Capsaicin i.c. significantly induced CGRP-ir in TG of the adult (AC vs. AS, p = 0.021). The capsaicin induced CGRP-ir of TG is suppressed by VPA in the adult group, borderline at 30 mg/kg (ACV30 vs. AC, p = 0.05), and significant at 100 mg/kg (ACV100 vs. AC, p = 0.034). (F) The capsaicin induced CGRP-ir is not increased compared to sham in pediatric group (PC vs. PS). H-stat, p > 0.05. (G) The immunochemical stain of dura shows linear CGRP-ir (arrow) along the meningeal vessels (arrowhead) at various dosages of VPA in both age groups. (H) Capsaicin i.c. significantly depletes CGRP-ir in the dura of adult (AC vs. AS, p = 0.034). The depletion of CGRP is significantly reversed by VPA at 30 mg/kg (ACV30 vs. AC, p = 0.034) and 100 mg/kg (ACV100 vs. AC, p = 0.014) in adult group. (I) Capsaicin i.c. significantly depletes CGRP-ir in the dura of pediatric (PC vs. PS, p = 0.021). The rescue of CGRP depletion is not obvious at 30 (PCV30 vs. PC) and 100 mg/kg (PCV100 vs. PC) in pediatric group. n = 4 in each group, ∗ p
    Figure Legend Snippet: c-Fos-ir in trigeminocervical complex (TCC) and CGRP-ir in trigeminal ganglia (TG) and dura of adult and pediatric rats treated with valproic acid (VPA). (A) The immunochemical stain of c-Fos shows c-Fos-ir at various dosages in both age groups. (B) Intracisternal capsaicin ( i.c. ) significantly induced c-Fos-ir in TCC of the adult compared to sham (AC vs. AS, p = 0.021). The capsaicin induced c-Fos-ir is not significantly suppressed by VPA in adult at either 30 (ACV30 vs. AC) or 100 (ACV100 vs. AC) mg/kg. (C) Capsaicin i.c. significantly induced c-Fos-ir in TCC of the pediatric (PC vs. PS, p = 0.021). The capsaicin induced c-Fos-ir is significantly suppressed by VPA in pediatric at 100 mg/kg (PCV100 vs. PC, p = 0.021) but not 30 mg/kg (PCV30 vs. PC). (D) The immunochemical stain of TG shows CGRP-ir at various dosages in both age groups. (E) Capsaicin i.c. significantly induced CGRP-ir in TG of the adult (AC vs. AS, p = 0.021). The capsaicin induced CGRP-ir of TG is suppressed by VPA in the adult group, borderline at 30 mg/kg (ACV30 vs. AC, p = 0.05), and significant at 100 mg/kg (ACV100 vs. AC, p = 0.034). (F) The capsaicin induced CGRP-ir is not increased compared to sham in pediatric group (PC vs. PS). H-stat, p > 0.05. (G) The immunochemical stain of dura shows linear CGRP-ir (arrow) along the meningeal vessels (arrowhead) at various dosages of VPA in both age groups. (H) Capsaicin i.c. significantly depletes CGRP-ir in the dura of adult (AC vs. AS, p = 0.034). The depletion of CGRP is significantly reversed by VPA at 30 mg/kg (ACV30 vs. AC, p = 0.034) and 100 mg/kg (ACV100 vs. AC, p = 0.014) in adult group. (I) Capsaicin i.c. significantly depletes CGRP-ir in the dura of pediatric (PC vs. PS, p = 0.021). The rescue of CGRP depletion is not obvious at 30 (PCV30 vs. PC) and 100 mg/kg (PCV100 vs. PC) in pediatric group. n = 4 in each group, ∗ p

    Techniques Used: Staining

    26) Product Images from "Identification of Urocortin 3 afferent projection to the ventromedial nucleus of hypothalamus in rat brain"

    Article Title: Identification of Urocortin 3 afferent projection to the ventromedial nucleus of hypothalamus in rat brain

    Journal: The Journal of comparative neurology

    doi: 10.1002/cne.22620

    Fluorescent confocal images showing Ucn 3 (A) and Enk (B) immunostaining in the VMH. The location of the images was indicated by the red box in D. C: Merged image of A and B showing no significant colocalization of Ucn 3 and Enk in the VMH. E–G: High magnification of the dorsomedial part of the VMH showing the staining of Ucn 3 (E) and Enk (F). Merged image (G) of E and F further illustrates little colocalization of the two neurosubstrates in this brain area. 3V: Third ventricle, ARH: Arcuate nucleus of hypothalamus, DMH: Dorsomedial nucleus of hypothalamus, f: Fornix, ic: Internal capsule, mt: Mammillothalamic tract, opt: Optic tract, VMHdm and VMHvl: Dorsomedial part (VMHdm) and ventrolateral part (VMHvl) of the ventromedial nucleus of hypothalamus. Scale bar: 50 μm (A), 20 μm (E).
    Figure Legend Snippet: Fluorescent confocal images showing Ucn 3 (A) and Enk (B) immunostaining in the VMH. The location of the images was indicated by the red box in D. C: Merged image of A and B showing no significant colocalization of Ucn 3 and Enk in the VMH. E–G: High magnification of the dorsomedial part of the VMH showing the staining of Ucn 3 (E) and Enk (F). Merged image (G) of E and F further illustrates little colocalization of the two neurosubstrates in this brain area. 3V: Third ventricle, ARH: Arcuate nucleus of hypothalamus, DMH: Dorsomedial nucleus of hypothalamus, f: Fornix, ic: Internal capsule, mt: Mammillothalamic tract, opt: Optic tract, VMHdm and VMHvl: Dorsomedial part (VMHdm) and ventrolateral part (VMHvl) of the ventromedial nucleus of hypothalamus. Scale bar: 50 μm (A), 20 μm (E).

    Techniques Used: Immunostaining, Staining

    Fluoresecnt confocal images showing Ucn 3 (A) and Enk (B) in the premammillary area indicated by the red box in D. C: Merged image of A and B indicates that Ucn 3 immunoreacitivty was found mostly in the PMV where as prominent Enk staining was observed in the PMD and thus no significant colocalization of Ucn 3 and Enk was found in these brain regions. 3V: Third ventricle, f: Fornix, ic: Internal capsule, LV: Lateral ventricle, mt: Mammillothalamic tract, PH: Posterior hypothalamus, PMD: Dorsal premammillary nucleus, PMV: Ventral premammillary nucleus. Scale bar: 50 μm.
    Figure Legend Snippet: Fluoresecnt confocal images showing Ucn 3 (A) and Enk (B) in the premammillary area indicated by the red box in D. C: Merged image of A and B indicates that Ucn 3 immunoreacitivty was found mostly in the PMV where as prominent Enk staining was observed in the PMD and thus no significant colocalization of Ucn 3 and Enk was found in these brain regions. 3V: Third ventricle, f: Fornix, ic: Internal capsule, LV: Lateral ventricle, mt: Mammillothalamic tract, PH: Posterior hypothalamus, PMD: Dorsal premammillary nucleus, PMV: Ventral premammillary nucleus. Scale bar: 50 μm.

    Techniques Used: Staining

    Organization of Ucn 3 projections to the VMH and LS. The magnitude of each pathway is roughly proportional to the thickness of the line representing it. Ucn 3 neurons in the PVHap/pBNST provide the major Ucn 3 afferent input to the VMH. Ucn 3 cells in the rPFH immediately caudal to the PVHap/pBNST Ucn 3 cell group project predominately to the LS. Ucn 3 cells in the MeA provide moderate afferent into the VMH. BNST: Bed nucleus of stria terminalis, f: Fornix, LS: Lateral septal nucleus, MeA: Medial amygdala, MePO: Median preoptic nucleus, PVHap: Anterior parvicellular part of the paraventricular nucleus of hypothalamus, VMH: Ventromedial nucleus of hypothalamus.
    Figure Legend Snippet: Organization of Ucn 3 projections to the VMH and LS. The magnitude of each pathway is roughly proportional to the thickness of the line representing it. Ucn 3 neurons in the PVHap/pBNST provide the major Ucn 3 afferent input to the VMH. Ucn 3 cells in the rPFH immediately caudal to the PVHap/pBNST Ucn 3 cell group project predominately to the LS. Ucn 3 cells in the MeA provide moderate afferent into the VMH. BNST: Bed nucleus of stria terminalis, f: Fornix, LS: Lateral septal nucleus, MeA: Medial amygdala, MePO: Median preoptic nucleus, PVHap: Anterior parvicellular part of the paraventricular nucleus of hypothalamus, VMH: Ventromedial nucleus of hypothalamus.

    Techniques Used: Microelectrode Array

    Representative stacked confocal images showing double immunofluorescent labeling of Ucn 3 (A, D) and Enk (B, E) in the lateral septum. C, F: Merged images of A and B (C) and D and E (F) showing high degree of colocalization of the two neuropeptides in neuronal fibers and axonal terminals in this area. LS: Lateral septum, LV: Lateral ventricle, MS: Medial septum, Scale bar: 50 μm (A), 20 μm (D).
    Figure Legend Snippet: Representative stacked confocal images showing double immunofluorescent labeling of Ucn 3 (A, D) and Enk (B, E) in the lateral septum. C, F: Merged images of A and B (C) and D and E (F) showing high degree of colocalization of the two neuropeptides in neuronal fibers and axonal terminals in this area. LS: Lateral septum, LV: Lateral ventricle, MS: Medial septum, Scale bar: 50 μm (A), 20 μm (D).

    Techniques Used: Labeling, Mass Spectrometry

    Representative stacked confocal images showing Ucn 3 (A) and Enk (B) immunostaining in the perifornical area indicated in D (red box). C: Merged images of Ucn 3 (A) and Enk (B) showing high degree of colocalization of the two materials in neurons and fibers in this brain area. 3V: Third ventricle, AH: Anterior hypothalamus, f: Fornix, ic: Internal capsule, mt: Mammillothalamic tract, PVH: Paraventricular nucleus of hypothalamus. opt: Optic tract. Scale bar: 25 μm.
    Figure Legend Snippet: Representative stacked confocal images showing Ucn 3 (A) and Enk (B) immunostaining in the perifornical area indicated in D (red box). C: Merged images of Ucn 3 (A) and Enk (B) showing high degree of colocalization of the two materials in neurons and fibers in this brain area. 3V: Third ventricle, AH: Anterior hypothalamus, f: Fornix, ic: Internal capsule, mt: Mammillothalamic tract, PVH: Paraventricular nucleus of hypothalamus. opt: Optic tract. Scale bar: 25 μm.

    Techniques Used: Immunostaining

    a: Double-label immunofluorescent staining of Ucn 3 (green) and Enk (red) in the posterior part of the BNST (A), the PVHap (B) and in the medial amygdala (C). The location of each image is indicated by red box in the respective drawing. Note that both Ucn 3 and Enk-positive cells and fibers are evident in these areas, but double-label cells or fibers were rarely observed. A few double-labeled cells and fibers (indicated by yellow arrowheads) were observed in the PVHap (B). 3V: Third ventricle, f: Fornix, ic: Internal capsule, MPN: Medial preoptic nucleus, LV: Lateral ventricle, ox: Optic chiasm, MeA: Medial amygdala, pBNST: posterior part of the bed nucleus of stria terminalis, PVHap: Anterior parvicellular part of the paraventricular nucleus of hypothalamus, SCN: Suprachiasmatic nucleus of hypothalamus, SON: Supraoptic nucleus of hypothalamus, sm: Stria medullaris of thalamus, st: Stria terminalis. Scale bar: 50 μm.
    Figure Legend Snippet: a: Double-label immunofluorescent staining of Ucn 3 (green) and Enk (red) in the posterior part of the BNST (A), the PVHap (B) and in the medial amygdala (C). The location of each image is indicated by red box in the respective drawing. Note that both Ucn 3 and Enk-positive cells and fibers are evident in these areas, but double-label cells or fibers were rarely observed. A few double-labeled cells and fibers (indicated by yellow arrowheads) were observed in the PVHap (B). 3V: Third ventricle, f: Fornix, ic: Internal capsule, MPN: Medial preoptic nucleus, LV: Lateral ventricle, ox: Optic chiasm, MeA: Medial amygdala, pBNST: posterior part of the bed nucleus of stria terminalis, PVHap: Anterior parvicellular part of the paraventricular nucleus of hypothalamus, SCN: Suprachiasmatic nucleus of hypothalamus, SON: Supraoptic nucleus of hypothalamus, sm: Stria medullaris of thalamus, st: Stria terminalis. Scale bar: 50 μm.

    Techniques Used: Staining, Labeling, Microelectrode Array

    A,H: Photomicrographs showing FG-positive cells (dark brown color cells) and Ucn 3 mRNA signals (black dot clusters) in rostral (A) and caudal (H) MeA. B, E: High power magnification of boxed areas in A to show colocalization of FG immunoreactivity (brown color cells) and Ucn 3 mRNA signals (black dot clusters). C, D, F, G: Photomicrographs of B (C, D) and E (F, G) at different focal planes showing FG-positive cells (C, F) and Ucn 3 mRNA signals (silver grain clusters, D, G). I: High power magnification of boxed area in H showing colocalization of FG immunoreactivity and Ucn 3 mRNA signals. J,K: Photomicrographs of I at different focal planes showing FG-positive cells (J) and Ucn 3 mRNA signals (silver grain clusters, K). Representative double-labeled cells are indicated by red arrowhead. Single-labeled FG-positive cells are indicated by blue arrowheads and single-labeled Ucn 3 mRNA-positive cells were indicated by yellow arrowheads. MeA: Medial amygdala, opt: Optic tract. Scale bar: 25 μm.
    Figure Legend Snippet: A,H: Photomicrographs showing FG-positive cells (dark brown color cells) and Ucn 3 mRNA signals (black dot clusters) in rostral (A) and caudal (H) MeA. B, E: High power magnification of boxed areas in A to show colocalization of FG immunoreactivity (brown color cells) and Ucn 3 mRNA signals (black dot clusters). C, D, F, G: Photomicrographs of B (C, D) and E (F, G) at different focal planes showing FG-positive cells (C, F) and Ucn 3 mRNA signals (silver grain clusters, D, G). I: High power magnification of boxed area in H showing colocalization of FG immunoreactivity and Ucn 3 mRNA signals. J,K: Photomicrographs of I at different focal planes showing FG-positive cells (J) and Ucn 3 mRNA signals (silver grain clusters, K). Representative double-labeled cells are indicated by red arrowhead. Single-labeled FG-positive cells are indicated by blue arrowheads and single-labeled Ucn 3 mRNA-positive cells were indicated by yellow arrowheads. MeA: Medial amygdala, opt: Optic tract. Scale bar: 25 μm.

    Techniques Used: Microelectrode Array, Labeling

    A: Bright-field photomicrograph showing double-labeling of FG-positive cells (dark brown cells) and Ucn 3 mRNA signals (black dot clusters) in the rostral perifornical hypothalamic area. B: High magnification photomicrograph of boxed area in A showing cells double-labeled with FG and Ucn 3 mRNA signals. C,D: Photomicrographs of B at different focal planes to show FG-positive cells (C) and Ucn 3 mRNA signals (silver grain clusters, D). Representative double-labeled cells are indicated by red arrowheads. FG single-labeled cells are indicated by blue arrowheads and Ucn 3 mRNA single-labeled cells are indicated by yellow arrowheads. f: Fornix. Scale bar: 50 μm.
    Figure Legend Snippet: A: Bright-field photomicrograph showing double-labeling of FG-positive cells (dark brown cells) and Ucn 3 mRNA signals (black dot clusters) in the rostral perifornical hypothalamic area. B: High magnification photomicrograph of boxed area in A showing cells double-labeled with FG and Ucn 3 mRNA signals. C,D: Photomicrographs of B at different focal planes to show FG-positive cells (C) and Ucn 3 mRNA signals (silver grain clusters, D). Representative double-labeled cells are indicated by red arrowheads. FG single-labeled cells are indicated by blue arrowheads and Ucn 3 mRNA single-labeled cells are indicated by yellow arrowheads. f: Fornix. Scale bar: 50 μm.

    Techniques Used: Labeling

    A: Bright-field photomicrograph showing FG-positive cells (dark brown cells) and Ucn 3 mRNA-positive signals (black dot clusters) in the posterior part of the BNST. B: High magnification of boxed area in A. Note that no colocalization of FG and Ucn 3 mRNA signals in this area. C, D: Photomicrographs of B at different focal planes to show FG-positive cells (C) and Ucn 3 mRNA signals (silver grain clusters, D). Representative single-label FG cells were indicated by blue arrowheads (C) and single-label Ucn 3 mRNA signals were indicated by yellow arrowheads. f: Fornix, pBNST: Posterior part of the bed nucleus of stria terminalis, sm: Stria medullaris of thalamus. Scale bar: 50 μm.
    Figure Legend Snippet: A: Bright-field photomicrograph showing FG-positive cells (dark brown cells) and Ucn 3 mRNA-positive signals (black dot clusters) in the posterior part of the BNST. B: High magnification of boxed area in A. Note that no colocalization of FG and Ucn 3 mRNA signals in this area. C, D: Photomicrographs of B at different focal planes to show FG-positive cells (C) and Ucn 3 mRNA signals (silver grain clusters, D). Representative single-label FG cells were indicated by blue arrowheads (C) and single-label Ucn 3 mRNA signals were indicated by yellow arrowheads. f: Fornix, pBNST: Posterior part of the bed nucleus of stria terminalis, sm: Stria medullaris of thalamus. Scale bar: 50 μm.

    Techniques Used:

    Schematic drawings of 25 μm coronal sections showing the distribution of FG-ir cells, Ucn 3 mRNA-positive cells and FG/Ucn 3 double-labeled cells in Ucn 3 expressing areas and surrounding regions. Each symbol for Ucn 3 (●) and for FG/Ucn 3 ( ) represents approximately two labeled cells and each symbol for FG ( ) represents approximately 8 cells.
    Figure Legend Snippet: Schematic drawings of 25 μm coronal sections showing the distribution of FG-ir cells, Ucn 3 mRNA-positive cells and FG/Ucn 3 double-labeled cells in Ucn 3 expressing areas and surrounding regions. Each symbol for Ucn 3 (●) and for FG/Ucn 3 ( ) represents approximately two labeled cells and each symbol for FG ( ) represents approximately 8 cells.

    Techniques Used: Labeling, Expressing

    A: Representative photomicrograph showing FG-positive cells (dark brown color cells) and Ucn 3 mRNA-positive signals (black dot clusters) in the PVHap. B: High power magnification of boxed area in A showing colocalization of FG immunoreactivity and Ucn 3 mRNA signals. C,D: Photomicrographs of B at different focal planes showing FG-positive cells (C) and Ucn 3 mRNA signals (silver grain clusters, D). Representative double-labeled cells are indicated by red arrowheads. Representative single-labeled FG-positive cells in C are indicated by blue arrowheads and single-labeled Ucn 3 mRNA-positive cells in D are indicated by yellow arrowheads. 3V: Third ventricle, f: Fornix,, PVHap: anterior parvicellular part of the paraventricular nucleus of hypothalamus. Scale bar: 50 μm.
    Figure Legend Snippet: A: Representative photomicrograph showing FG-positive cells (dark brown color cells) and Ucn 3 mRNA-positive signals (black dot clusters) in the PVHap. B: High power magnification of boxed area in A showing colocalization of FG immunoreactivity and Ucn 3 mRNA signals. C,D: Photomicrographs of B at different focal planes showing FG-positive cells (C) and Ucn 3 mRNA signals (silver grain clusters, D). Representative double-labeled cells are indicated by red arrowheads. Representative single-labeled FG-positive cells in C are indicated by blue arrowheads and single-labeled Ucn 3 mRNA-positive cells in D are indicated by yellow arrowheads. 3V: Third ventricle, f: Fornix,, PVHap: anterior parvicellular part of the paraventricular nucleus of hypothalamus. Scale bar: 50 μm.

    Techniques Used: Labeling

    A. Bright-field photomicrograph showing FG-positive cells (dark brown color cells) and Ucn 3 mRNA-positive signals (black dot clusters) in the posterior part of the BNST. B,E: High power magnification of boxed areas in A showing colocalization of FG immunoreactivity and Ucn 3 mRNA signals. C, D, F, G: Photomicrographs of B (C, D) and E (F, G) at different focal planes showing FG-positive cells (C, F) and Ucn 3 mRNA signals (silver grain clusters, D, G). Representative double-labeled cells are indicated by red arrowheads. Representative single-labeled FG-positive cells in C and F are indicated by blue arrowheads and representative single-labeled Ucn 3 mRNA-positive cells in D and G are indicated by yellow arrowheads. f: Fornix, sm: Stria medullaris of thalamus. Scale bar: 25 μm.
    Figure Legend Snippet: A. Bright-field photomicrograph showing FG-positive cells (dark brown color cells) and Ucn 3 mRNA-positive signals (black dot clusters) in the posterior part of the BNST. B,E: High power magnification of boxed areas in A showing colocalization of FG immunoreactivity and Ucn 3 mRNA signals. C, D, F, G: Photomicrographs of B (C, D) and E (F, G) at different focal planes showing FG-positive cells (C, F) and Ucn 3 mRNA signals (silver grain clusters, D, G). Representative double-labeled cells are indicated by red arrowheads. Representative single-labeled FG-positive cells in C and F are indicated by blue arrowheads and representative single-labeled Ucn 3 mRNA-positive cells in D and G are indicated by yellow arrowheads. f: Fornix, sm: Stria medullaris of thalamus. Scale bar: 25 μm.

    Techniques Used: Labeling

    A: Fluorescent image showing FG tracer injection site (white arrow) in the septal region. The injection site centers near the intermediate part of the lateral septal nucleus (B, hatched area), which receives a prominent Ucn 3 fiber innervation. cc: Corpus callosum, LSd: Dorsal part of the lateral septal nucleus, LSi: Intermediate part of the lateral septal nucleus, LSv: Ventral part of the lateral septal nucleus, LV: Lateral ventricle, MS: Medial septal nucleus. Scale bar: 100 μm.
    Figure Legend Snippet: A: Fluorescent image showing FG tracer injection site (white arrow) in the septal region. The injection site centers near the intermediate part of the lateral septal nucleus (B, hatched area), which receives a prominent Ucn 3 fiber innervation. cc: Corpus callosum, LSd: Dorsal part of the lateral septal nucleus, LSi: Intermediate part of the lateral septal nucleus, LSv: Ventral part of the lateral septal nucleus, LV: Lateral ventricle, MS: Medial septal nucleus. Scale bar: 100 μm.

    Techniques Used: Injection, Mass Spectrometry

    27) Product Images from "Hypoxia promotes fibrogenesis in vivo via HIF-1 stimulation of epithelial-to-mesenchymal transition"

    Article Title: Hypoxia promotes fibrogenesis in vivo via HIF-1 stimulation of epithelial-to-mesenchymal transition

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI30487

    Inhibition of lysyl oxidases reduces fibrosis in UUO kidneys. Shown are the number of FSP-1–positive cells (top panel) and the area stained for collagen by sirius red (bottom panel) in vehicle-treated (VEH-UUO) and BAPN-treated UUO (BAPN-UUO) kidneys; original magnification, ×100 (top panel) and ×400 (bottom panel). Scale bars represent mean values ± SEM; * P
    Figure Legend Snippet: Inhibition of lysyl oxidases reduces fibrosis in UUO kidneys. Shown are the number of FSP-1–positive cells (top panel) and the area stained for collagen by sirius red (bottom panel) in vehicle-treated (VEH-UUO) and BAPN-treated UUO (BAPN-UUO) kidneys; original magnification, ×100 (top panel) and ×400 (bottom panel). Scale bars represent mean values ± SEM; * P

    Techniques Used: Inhibition, Staining

    Hif-1 promotes EMT in PTECs. ( A ) Upper-left panel: Schematic illustrating the genetic make-up of mice used for the in vitro EMT studies. Mice expressed the ROSA26RLacZ reporter (top) in conjunction with the PEPCK-cre transgene, in the presence (+) or absence (–) of the floxed Hif1a conditional allele (bottom). Triangles indicate the presence of loxP sites. Hif1a +/+ or Hif1a –/– PTECs were cultured under normoxia or hypoxia for 0–5 days. Cells were stained for β-gal (red) and the mesenchymal marker FSP-1 (green; original magnification, ×400). Epithelial cells undergoing EMT stained both red and green (arrows). ( B ) Percent FSP-1–positive epithelial cells in Hif1a +/+ or Hif1a –/– cultures exposed to normoxia (N) or hypoxia (H) for 1–5 days. Scale bars represent mean values ± SEM. * P
    Figure Legend Snippet: Hif-1 promotes EMT in PTECs. ( A ) Upper-left panel: Schematic illustrating the genetic make-up of mice used for the in vitro EMT studies. Mice expressed the ROSA26RLacZ reporter (top) in conjunction with the PEPCK-cre transgene, in the presence (+) or absence (–) of the floxed Hif1a conditional allele (bottom). Triangles indicate the presence of loxP sites. Hif1a +/+ or Hif1a –/– PTECs were cultured under normoxia or hypoxia for 0–5 days. Cells were stained for β-gal (red) and the mesenchymal marker FSP-1 (green; original magnification, ×400). Epithelial cells undergoing EMT stained both red and green (arrows). ( B ) Percent FSP-1–positive epithelial cells in Hif1a +/+ or Hif1a –/– cultures exposed to normoxia (N) or hypoxia (H) for 1–5 days. Scale bars represent mean values ± SEM. * P

    Techniques Used: Mouse Assay, In Vitro, Cell Culture, Staining, Marker

    Deletion of Hif1a in PTECs attenuates renal fibrogenesis. Hif1a +/+ and Hif1a –/– kidneys were stained for collagen content (sirius red staining of collagen fibers shown in red; original magnification, ×200; n = 8 for mutant and n = 7 for control), macrophage marker F4/80 (original magnification, ×400; n = 3 in each group), and EMT marker FSP-1 (original magnification, ×400; n = 9 for mutant and n = 5 for control). For statistical analysis, sirius red–positive areas from 10 individual measurements per mouse were averaged across control and mutant cohorts. Morphometric analysis showed a reduction of all 3 stains in Hif1a mutant tissues. Scale bars represent mean values ± SEM; * P
    Figure Legend Snippet: Deletion of Hif1a in PTECs attenuates renal fibrogenesis. Hif1a +/+ and Hif1a –/– kidneys were stained for collagen content (sirius red staining of collagen fibers shown in red; original magnification, ×200; n = 8 for mutant and n = 7 for control), macrophage marker F4/80 (original magnification, ×400; n = 3 in each group), and EMT marker FSP-1 (original magnification, ×400; n = 9 for mutant and n = 5 for control). For statistical analysis, sirius red–positive areas from 10 individual measurements per mouse were averaged across control and mutant cohorts. Morphometric analysis showed a reduction of all 3 stains in Hif1a mutant tissues. Scale bars represent mean values ± SEM; * P

    Techniques Used: Staining, Mutagenesis, Marker

    28) Product Images from "PARP Inhibitor Activity Correlates with SLFN11 Expression and Demonstrates Synergy with Temozolomide in Small Cell Lung Cancer"

    Article Title: PARP Inhibitor Activity Correlates with SLFN11 Expression and Demonstrates Synergy with Temozolomide in Small Cell Lung Cancer

    Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

    doi: 10.1158/1078-0432.CCR-16-1040

    SLFN11 protein expression correlates with talazoparib efficacy in patient-derived xenografts and SLFN11 mRNA is expressed bimodally in primary patient samples (A) Representative images of immunohistochemical staining against SLFN11 for all tested PDXs are shown. The H-score and modified H-score (H mod ) for SLFN11 nuclear staining for each PDX model is displayed. Scale bar, 50 μm. (B) Percent change in tumor volume at end of study for each individual animal and displayed in order of SLFN11 H-score. End of study difference in tumor size between vehicle and treatment groups were significant for JHU-LX22, JHU-LX110, and SCRX-Lu149 (p = 0.0286, p = 0.0286, p = 0.0079, respectively) P -values by the Wilcoxon-Mann-Whitney test. (C) Percent tumor growth inhibition for each PDX model. Mean ± SD shown. The delta method was used to compute the variance used for SD calculations. (D) SLFN11 gene expression of primary SCLC samples plotted with publically available datasets from The Cancer Genome Atlas (TCGA) of other histologies are displayed here. The inset displays a bimodal distribution of SLFN11 gene expression ( blue dashed line ). Median ± SD shown.
    Figure Legend Snippet: SLFN11 protein expression correlates with talazoparib efficacy in patient-derived xenografts and SLFN11 mRNA is expressed bimodally in primary patient samples (A) Representative images of immunohistochemical staining against SLFN11 for all tested PDXs are shown. The H-score and modified H-score (H mod ) for SLFN11 nuclear staining for each PDX model is displayed. Scale bar, 50 μm. (B) Percent change in tumor volume at end of study for each individual animal and displayed in order of SLFN11 H-score. End of study difference in tumor size between vehicle and treatment groups were significant for JHU-LX22, JHU-LX110, and SCRX-Lu149 (p = 0.0286, p = 0.0286, p = 0.0079, respectively) P -values by the Wilcoxon-Mann-Whitney test. (C) Percent tumor growth inhibition for each PDX model. Mean ± SD shown. The delta method was used to compute the variance used for SD calculations. (D) SLFN11 gene expression of primary SCLC samples plotted with publically available datasets from The Cancer Genome Atlas (TCGA) of other histologies are displayed here. The inset displays a bimodal distribution of SLFN11 gene expression ( blue dashed line ). Median ± SD shown.

    Techniques Used: Expressing, Derivative Assay, Immunohistochemistry, Staining, Modification, MANN-WHITNEY, Inhibition

    29) Product Images from "DEDD regulates degradation of intermediate filaments during apoptosis"

    Article Title: DEDD regulates degradation of intermediate filaments during apoptosis

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200112124

    Caspase-3 is recruited to IFs and associates with DEDD. (A) MCF7 cells were transiently transfected with procaspase-3–GFP, treated with STS (0, 2 and 4 h) and stained for K8. Cells were analyzed by confocal microscopy. Shown are two-dimensional pictures of three- dimensional images, which were created using 20 overlaying 0.5 μm z-sections. Merge, overlay of GFP fluorescence and K8 staining. Arrowheads denote caspase-3–GFP positivity on IFs. A three-dimensional image of this panel is depicted in Video 1 (available online at http://www.jcb.org/cgi/content/full/jcb.200112124/DC1 ). Bar, 10 μm. (B) Immunoprecipitation of caspase-3 from MCF7-C3 (with a rabbit polyclonal caspase-3 antibody) coimmunoprecipitated DEDD 2Ub as detected by biotinylated PRO29 and anti-ubiquitin mAb (left two panels). Control blot (right) with a different caspase-3 (CASP-3) mAb detected efficient caspase-3 immunoprecipitation. Note, the same blot was probed sequentially in the order PRO29, anti-CASP-3, anti-ubiquitin. All M r notations on left side of blots in kD.
    Figure Legend Snippet: Caspase-3 is recruited to IFs and associates with DEDD. (A) MCF7 cells were transiently transfected with procaspase-3–GFP, treated with STS (0, 2 and 4 h) and stained for K8. Cells were analyzed by confocal microscopy. Shown are two-dimensional pictures of three- dimensional images, which were created using 20 overlaying 0.5 μm z-sections. Merge, overlay of GFP fluorescence and K8 staining. Arrowheads denote caspase-3–GFP positivity on IFs. A three-dimensional image of this panel is depicted in Video 1 (available online at http://www.jcb.org/cgi/content/full/jcb.200112124/DC1 ). Bar, 10 μm. (B) Immunoprecipitation of caspase-3 from MCF7-C3 (with a rabbit polyclonal caspase-3 antibody) coimmunoprecipitated DEDD 2Ub as detected by biotinylated PRO29 and anti-ubiquitin mAb (left two panels). Control blot (right) with a different caspase-3 (CASP-3) mAb detected efficient caspase-3 immunoprecipitation. Note, the same blot was probed sequentially in the order PRO29, anti-CASP-3, anti-ubiquitin. All M r notations on left side of blots in kD.

    Techniques Used: Transfection, Staining, Confocal Microscopy, Fluorescence, Immunoprecipitation

    30) Product Images from "Skap2 is required for β2 integrin–mediated neutrophil recruitment and functions"

    Article Title: Skap2 is required for β2 integrin–mediated neutrophil recruitment and functions

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20160647

    Cross talk of Skap2 and WASp is indispensable for β 2 integrin activation and neutrophil recruitment. (A–C) IVM of TNF-inflamed postcapillary venules of WT, Was −/− , and Skap2 −/− Was −/− mice. Rolling velocities (A), number of adherent cells (B), and number of extravasated cells (C) 2 h after TNF application are shown. n = 4 mice/group. (D) Adhesion of neutrophils in postcapillary venules of WT, Was −/− , and Skap2 −/− Was −/− mice after i.v. injection of CXCL1. n = 4 mice/group. (E and F) Soluble ICAM-1 binding (E) and soluble fibrinogen binding (F) of CXCL1-stimulated WT, Was −/− , and Skap2 −/− Was −/− neutrophils. n = 3. (G) Immunoprecipitation of Skap2 in unstimulated WT neutrophils or neutrophils plated on E-selectin with shear or stimulated with CXCL1 in solution. Immunoprecipitates were immunoblotted with anti-WASp and anti-Skap2 antibody. Input was with anti–α-tubulin antibody. n = 3. (H) WT neutrophils were left unstimulated, plated on E-selectin with shear, or stimulated with CXCL1 in solution. Lysates were incubated with GST alone (control), GST fusion proteins of the Skap2 CC, PH, or SH3 domains, or full-length Skap2. Precipitates were immunoblotted with anti-WASp, and input was with anti–α-tubulin antibody. n = 3. (I and J) In vitro co-purification of His-WASp by different Skap2 GST fusion proteins. Precipitates were immunoblotted with anti-His or anti-GST, and input controls were with anti-GST antibody. n = 3. (K–N) WT and Skap2 −/− or Was −/− neutrophils were left unstimulated, plated on E-selectin with shear, or stimulated with CXCL1 in solution. Lysates were immunoprecipitated with anti-WASp (K and L) or anti-Skap2 (M and N) antibody followed by immunoblotting with anti-phosphotyrosine (4G10), anti-WASp, or anti-Skap2 antibody. Input was immunoblotted with anti–α-tubulin antibody. Quantification is shown on the right. n = 3. *, P
    Figure Legend Snippet: Cross talk of Skap2 and WASp is indispensable for β 2 integrin activation and neutrophil recruitment. (A–C) IVM of TNF-inflamed postcapillary venules of WT, Was −/− , and Skap2 −/− Was −/− mice. Rolling velocities (A), number of adherent cells (B), and number of extravasated cells (C) 2 h after TNF application are shown. n = 4 mice/group. (D) Adhesion of neutrophils in postcapillary venules of WT, Was −/− , and Skap2 −/− Was −/− mice after i.v. injection of CXCL1. n = 4 mice/group. (E and F) Soluble ICAM-1 binding (E) and soluble fibrinogen binding (F) of CXCL1-stimulated WT, Was −/− , and Skap2 −/− Was −/− neutrophils. n = 3. (G) Immunoprecipitation of Skap2 in unstimulated WT neutrophils or neutrophils plated on E-selectin with shear or stimulated with CXCL1 in solution. Immunoprecipitates were immunoblotted with anti-WASp and anti-Skap2 antibody. Input was with anti–α-tubulin antibody. n = 3. (H) WT neutrophils were left unstimulated, plated on E-selectin with shear, or stimulated with CXCL1 in solution. Lysates were incubated with GST alone (control), GST fusion proteins of the Skap2 CC, PH, or SH3 domains, or full-length Skap2. Precipitates were immunoblotted with anti-WASp, and input was with anti–α-tubulin antibody. n = 3. (I and J) In vitro co-purification of His-WASp by different Skap2 GST fusion proteins. Precipitates were immunoblotted with anti-His or anti-GST, and input controls were with anti-GST antibody. n = 3. (K–N) WT and Skap2 −/− or Was −/− neutrophils were left unstimulated, plated on E-selectin with shear, or stimulated with CXCL1 in solution. Lysates were immunoprecipitated with anti-WASp (K and L) or anti-Skap2 (M and N) antibody followed by immunoblotting with anti-phosphotyrosine (4G10), anti-WASp, or anti-Skap2 antibody. Input was immunoblotted with anti–α-tubulin antibody. Quantification is shown on the right. n = 3. *, P

    Techniques Used: Activation Assay, Mouse Assay, Injection, Binding Assay, Immunoprecipitation, Incubation, In Vitro, Copurification

    31) Product Images from "Ablation of BRaf Impairs Neuronal Differentiation in the Postnatal Hippocampus and Cerebellum"

    Article Title: Ablation of BRaf Impairs Neuronal Differentiation in the Postnatal Hippocampus and Cerebellum

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0058259

    Nestin-Cre mediated deletion of BRaf impairs formation of synaptic networks of cultured hippocampal neurons. (A) Cells from the hippocampi of newborn mice were cultured for 6 days in vitro, fixed and stained for expression of BRaf and Map2. Scale bar = 25 µm . (B) Quantification of BRaf-positive, Map2-positive cells as a fraction of DAPI-labelled cells isolated from hippocampi at P0/P1 of ctrl or cKO mice and grown for 6 days in vitro. Data are mean ±s.e.m.; n = 5.
    Figure Legend Snippet: Nestin-Cre mediated deletion of BRaf impairs formation of synaptic networks of cultured hippocampal neurons. (A) Cells from the hippocampi of newborn mice were cultured for 6 days in vitro, fixed and stained for expression of BRaf and Map2. Scale bar = 25 µm . (B) Quantification of BRaf-positive, Map2-positive cells as a fraction of DAPI-labelled cells isolated from hippocampi at P0/P1 of ctrl or cKO mice and grown for 6 days in vitro. Data are mean ±s.e.m.; n = 5.

    Techniques Used: Cell Culture, Mouse Assay, In Vitro, Staining, Expressing, Isolation

    32) Product Images from "Presenilin Binding Protein Is Associated with Neurofibrillary Alterations in Alzheimer's Disease and Stimulates Tau Phosphorylation"

    Article Title: Presenilin Binding Protein Is Associated with Neurofibrillary Alterations in Alzheimer's Disease and Stimulates Tau Phosphorylation

    Journal: The American Journal of Pathology

    doi:

    Immunocytochemical analysis of PBP immunoreactivity in human brain. A: Control PBP mildly immunolabeled the neuropil and occasionally the pyramidal cells. In the neuropil, some neuritic processes and presynaptic boutons were labeled. Scale bar, 15 μm. B and C: In AD frontal cortex and hippocampus, PBP antibody strongly immunolabeled the NFT ( arrowheads ), the neuropil threads, and a subpopulation of the neuritic component of the plaques. D: Preimmune serum labeled little material. E and F: Laser scanning confocal imaging. Sections were double labeled with an antibody against phosphorylated tau (AT8, green) and PBP (red). Scale bar, 15 μm. Colocalization of yellow and arrows of PBP and tau tangles ( E and F ), in the neuropil threads ( G ), fusiform neurites in the plaque ( H ).
    Figure Legend Snippet: Immunocytochemical analysis of PBP immunoreactivity in human brain. A: Control PBP mildly immunolabeled the neuropil and occasionally the pyramidal cells. In the neuropil, some neuritic processes and presynaptic boutons were labeled. Scale bar, 15 μm. B and C: In AD frontal cortex and hippocampus, PBP antibody strongly immunolabeled the NFT ( arrowheads ), the neuropil threads, and a subpopulation of the neuritic component of the plaques. D: Preimmune serum labeled little material. E and F: Laser scanning confocal imaging. Sections were double labeled with an antibody against phosphorylated tau (AT8, green) and PBP (red). Scale bar, 15 μm. Colocalization of yellow and arrows of PBP and tau tangles ( E and F ), in the neuropil threads ( G ), fusiform neurites in the plaque ( H ).

    Techniques Used: Immunolabeling, Labeling, Imaging

    33) Product Images from "Identification of lectin counter-receptors on cell membranes by proximity labeling"

    Article Title: Identification of lectin counter-receptors on cell membranes by proximity labeling

    Journal: Glycobiology

    doi: 10.1093/glycob/cwx063

    The binding and biotinylation activities of Sn-HRP-Fc chimeras. Sn3L and Sn0L denotes there are 3 and 0 linkers (GSGGGGSGGG) between the Sn and HRP respectively. SnR97A3L has a R97A mutation in Sn of the Sn3L chimera and was used as a negative control. For the binding assay, the concentration of each chimera was kept constant at 2.5 μg/mL, with addition of 7.5, 2.5 and 0.8 μg/mL FITC-conjugated goat anti-human IgG Fc to prepare immune-complexes at ratios of 3:1, 1:1 and 0.3:1 anti-Fc:Sn chimera, respectively, which are shown after the names of Sn chimeras in the figure. The binding to human erythrocytes was analyzed by flow cytometry. For the biotinylation assay, the concentration of each chimera was kept constant at 10 μg/mL, and complexes at ratios of 3:1, 1:1 and 0.3:1 anti-Fc-FITC:Sn-HRP-Fc chimera were prepared, which are shown after the names of Sn chimeras in the figure. ( A ) Binding of Sn3L to erythrocytes. ( B ) Binding of Sn0L to erythrocytes. ( C ) Biotinylation of erythrocytes by Sn chimeras. Cells were lysed and blotted with streptavidin-HRP. ( D ) Erythrocyte lysate blotted with anti-glycophorin A. ( E ) Total erythrocyte surface proteins labeled using sulfo-NHS-SS-biotin and the cell lysate was blotted by streptavidin-HRP. ( F ) Proteins biotinylated using Sn-HRP-Fc chimeras were pulled down with streptavidin magnetic beads, eluted by reducing LDS sample buffer and blotted with anti-glycophorin A. This figure is available in black and white in print and in color at Glycobiology online.
    Figure Legend Snippet: The binding and biotinylation activities of Sn-HRP-Fc chimeras. Sn3L and Sn0L denotes there are 3 and 0 linkers (GSGGGGSGGG) between the Sn and HRP respectively. SnR97A3L has a R97A mutation in Sn of the Sn3L chimera and was used as a negative control. For the binding assay, the concentration of each chimera was kept constant at 2.5 μg/mL, with addition of 7.5, 2.5 and 0.8 μg/mL FITC-conjugated goat anti-human IgG Fc to prepare immune-complexes at ratios of 3:1, 1:1 and 0.3:1 anti-Fc:Sn chimera, respectively, which are shown after the names of Sn chimeras in the figure. The binding to human erythrocytes was analyzed by flow cytometry. For the biotinylation assay, the concentration of each chimera was kept constant at 10 μg/mL, and complexes at ratios of 3:1, 1:1 and 0.3:1 anti-Fc-FITC:Sn-HRP-Fc chimera were prepared, which are shown after the names of Sn chimeras in the figure. ( A ) Binding of Sn3L to erythrocytes. ( B ) Binding of Sn0L to erythrocytes. ( C ) Biotinylation of erythrocytes by Sn chimeras. Cells were lysed and blotted with streptavidin-HRP. ( D ) Erythrocyte lysate blotted with anti-glycophorin A. ( E ) Total erythrocyte surface proteins labeled using sulfo-NHS-SS-biotin and the cell lysate was blotted by streptavidin-HRP. ( F ) Proteins biotinylated using Sn-HRP-Fc chimeras were pulled down with streptavidin magnetic beads, eluted by reducing LDS sample buffer and blotted with anti-glycophorin A. This figure is available in black and white in print and in color at Glycobiology online.

    Techniques Used: Binding Assay, Mutagenesis, Negative Control, Concentration Assay, Flow Cytometry, Cytometry, Cell Surface Biotinylation Assay, Labeling, Magnetic Beads

    34) Product Images from "Apolipoprotein E4 Produced in GABAergic Interneurons Causes Learning and Memory Deficits in Mice"

    Article Title: Apolipoprotein E4 Produced in GABAergic Interneurons Causes Learning and Memory Deficits in Mice

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.2281-14.2014

    Generation and characterization of apoE-fKI/Dlx-Cre mice. A , B , Expression of ZsGreen1 (green) in the cortex and hippocampus of Dlx-Cre-positive ( A ) and -negative ( B ) ZsGreen1 reporter mice. Scale bars: 250 μm. C–F , ZsGreen1 was expressed in GABA-positive ( C , E ) and somatostatin (SOM)-positive ( D , F ) inhibitory interneurons in the cortex and hippocampus of Dlx-Cre-positive apoE-fKI mice. Scale bars: 15 μm. G , H , Anti-apoE immunostaining revealed the presence of apoE protein in GABA-positive hilar interneurons in aged apoE4-fKI mice ( G ), but not in apoE4-fKI/Dlx-Cre mice ( H ). Scale bars: 15 μm. I , Images of GABA-positive hilar interneurons before and after laser capture. Inhibitory interneuron were identified by anti-GABA immunostaining. Arrows indicate the cell before and after laser capture. Scale bars: 15 μm. J , Schematic of primers and their binding sites on the human APOE gene. K , PCR with primers 1 and 2 resulted in an amplified product in samples of apoE4-fKI mice, but not in samples of apoE4-fKI/Dlx-Cre mice (left). PCR with primers 1 and 3 resulted in an amplified product in samples of apoE4-fKI/Dlx-Cre mice, but not in samples of apoE4-fKI mice (right). Fifty nuclei per sample were used. L , Representative fluorescent Western blot of apoE (green) and GAPDH (red) in cortical and hippocampal lysates of 17-month-old female mice with different genotypes. M , N , Quantification of apoE protein levels relative to GAPDH protein levels in cortical ( M ) and hippocampal lysates ( N ) of 17-month-old mice ( n = 5 per genotype). For both the cortex and the hippocampus, the apoE level in apoE3-fKI mice was normalized to 1, and apoE levels in other groups of mice were presented relative to those in apoE3-fKI mice. * p
    Figure Legend Snippet: Generation and characterization of apoE-fKI/Dlx-Cre mice. A , B , Expression of ZsGreen1 (green) in the cortex and hippocampus of Dlx-Cre-positive ( A ) and -negative ( B ) ZsGreen1 reporter mice. Scale bars: 250 μm. C–F , ZsGreen1 was expressed in GABA-positive ( C , E ) and somatostatin (SOM)-positive ( D , F ) inhibitory interneurons in the cortex and hippocampus of Dlx-Cre-positive apoE-fKI mice. Scale bars: 15 μm. G , H , Anti-apoE immunostaining revealed the presence of apoE protein in GABA-positive hilar interneurons in aged apoE4-fKI mice ( G ), but not in apoE4-fKI/Dlx-Cre mice ( H ). Scale bars: 15 μm. I , Images of GABA-positive hilar interneurons before and after laser capture. Inhibitory interneuron were identified by anti-GABA immunostaining. Arrows indicate the cell before and after laser capture. Scale bars: 15 μm. J , Schematic of primers and their binding sites on the human APOE gene. K , PCR with primers 1 and 2 resulted in an amplified product in samples of apoE4-fKI mice, but not in samples of apoE4-fKI/Dlx-Cre mice (left). PCR with primers 1 and 3 resulted in an amplified product in samples of apoE4-fKI/Dlx-Cre mice, but not in samples of apoE4-fKI mice (right). Fifty nuclei per sample were used. L , Representative fluorescent Western blot of apoE (green) and GAPDH (red) in cortical and hippocampal lysates of 17-month-old female mice with different genotypes. M , N , Quantification of apoE protein levels relative to GAPDH protein levels in cortical ( M ) and hippocampal lysates ( N ) of 17-month-old mice ( n = 5 per genotype). For both the cortex and the hippocampus, the apoE level in apoE3-fKI mice was normalized to 1, and apoE levels in other groups of mice were presented relative to those in apoE3-fKI mice. * p

    Techniques Used: Mouse Assay, Expressing, Immunostaining, Binding Assay, Polymerase Chain Reaction, Amplification, Western Blot

    Generation and characterization of apoE-fKI/Syn-1-Cre mice. A , B , Expression of ZsGreen1 (green) in the cortex and hippocampus of Syn-1-Cre-positive ( A ) and -negative ( B ) ZsGreen1 reporter mice. Scale bars: 250 μm. C–H , ZsGreen1 was expressed in NeuN-positive neurons ( C , F ) and GABA-positive inhibitory interneurons ( E , H ), but not in GFAP-positive astrocytes ( D , G ) in the cortex and hippocampus of Syn-1-Cre-positive apoE-fKI mice. Scale bars: 30 μm. I , Images of the dentate granular cell layer before and after laser capture. Neuronal nuclei were identified by anti-NeuN immunostaining. Arrows indicate the cell before and after laser capture. Scale bars: 30 μm. J , Schematic of primers and their binding sites on the human APOE gene. K , PCR with primers 1 and 2 resulted in an amplified product in samples of apoE4-fKI mice, but not in samples of apoE4-fKI/Syn-1-Cre mice (left). PCR with primers 1 and 3 resulted in an amplified product in samples of apoE4-fKI/Syn-1-Cre mice, but not in samples of apoE4-fKI mice (right). Fifty nuclei per sample were used. L , Representative fluorescent Western blot of apoE (green) and GAPDH (red) in cortical and hippocampal lysates of 17-month-old female mice with different genotypes. M , N , Quantification of apoE protein levels relative to GAPDH protein levels in cortical ( M ) and hippocampal lysates ( N ) of 17-month-old mice ( n = 5 per genotype). ApoE levels in apoE3-fKI mice were normalized to 1, and apoE levels in other groups of mice were presented relative to those in apoE3-fKI mice. * p
    Figure Legend Snippet: Generation and characterization of apoE-fKI/Syn-1-Cre mice. A , B , Expression of ZsGreen1 (green) in the cortex and hippocampus of Syn-1-Cre-positive ( A ) and -negative ( B ) ZsGreen1 reporter mice. Scale bars: 250 μm. C–H , ZsGreen1 was expressed in NeuN-positive neurons ( C , F ) and GABA-positive inhibitory interneurons ( E , H ), but not in GFAP-positive astrocytes ( D , G ) in the cortex and hippocampus of Syn-1-Cre-positive apoE-fKI mice. Scale bars: 30 μm. I , Images of the dentate granular cell layer before and after laser capture. Neuronal nuclei were identified by anti-NeuN immunostaining. Arrows indicate the cell before and after laser capture. Scale bars: 30 μm. J , Schematic of primers and their binding sites on the human APOE gene. K , PCR with primers 1 and 2 resulted in an amplified product in samples of apoE4-fKI mice, but not in samples of apoE4-fKI/Syn-1-Cre mice (left). PCR with primers 1 and 3 resulted in an amplified product in samples of apoE4-fKI/Syn-1-Cre mice, but not in samples of apoE4-fKI mice (right). Fifty nuclei per sample were used. L , Representative fluorescent Western blot of apoE (green) and GAPDH (red) in cortical and hippocampal lysates of 17-month-old female mice with different genotypes. M , N , Quantification of apoE protein levels relative to GAPDH protein levels in cortical ( M ) and hippocampal lysates ( N ) of 17-month-old mice ( n = 5 per genotype). ApoE levels in apoE3-fKI mice were normalized to 1, and apoE levels in other groups of mice were presented relative to those in apoE3-fKI mice. * p

    Techniques Used: Mouse Assay, Expressing, Immunostaining, Binding Assay, Polymerase Chain Reaction, Amplification, Western Blot

    Generation and characterization of apoE-fKI/GFAP-Cre mice. A , B , Representative images of fluorescent immunostaining with anti-Cre recombinase (green) and anti-GFAP (red) in the cortex ( A ) and hippocampus ( B ) of apoE-fKI/GFAP-Cre mice. C , D , Neurons immunostained with anti-NeuN (red) did not express Cre-recombinase in the cortex ( C ) or hippocampus ( D ) of apoE-fKI/GFAP-Cre mice. E , F , Anti-apoE (green) and anti-GFAP (red) double immunostaining revealed that apoE expression was dramatically decreased in cortical ( E ) and hippocampal ( F ) astrocytes in apoE3-fKI/GFAP-Cre and apoE4-fKI/GFAP-Cre mice. G , Representative fluorescent Western blot of apoE (green) and GAPDH (red) in cortical and hippocampal lysates of 17-month-old female mice with different genotypes. H , I , Quantification of apoE protein levels relative to GAPDH in cortical ( H ) and hippocampal lysates ( I ) of 17-month-old mice ( n = 5/genotype). ApoE levels in apoE3-fKI mice were normalized to 1, and apoE levels in other groups of mice were presented relative to those in apoE3-fKI mice. *** p
    Figure Legend Snippet: Generation and characterization of apoE-fKI/GFAP-Cre mice. A , B , Representative images of fluorescent immunostaining with anti-Cre recombinase (green) and anti-GFAP (red) in the cortex ( A ) and hippocampus ( B ) of apoE-fKI/GFAP-Cre mice. C , D , Neurons immunostained with anti-NeuN (red) did not express Cre-recombinase in the cortex ( C ) or hippocampus ( D ) of apoE-fKI/GFAP-Cre mice. E , F , Anti-apoE (green) and anti-GFAP (red) double immunostaining revealed that apoE expression was dramatically decreased in cortical ( E ) and hippocampal ( F ) astrocytes in apoE3-fKI/GFAP-Cre and apoE4-fKI/GFAP-Cre mice. G , Representative fluorescent Western blot of apoE (green) and GAPDH (red) in cortical and hippocampal lysates of 17-month-old female mice with different genotypes. H , I , Quantification of apoE protein levels relative to GAPDH in cortical ( H ) and hippocampal lysates ( I ) of 17-month-old mice ( n = 5/genotype). ApoE levels in apoE3-fKI mice were normalized to 1, and apoE levels in other groups of mice were presented relative to those in apoE3-fKI mice. *** p

    Techniques Used: Mouse Assay, Immunostaining, Double Immunostaining, Expressing, Western Blot

    35) Product Images from "Intestinal tumorigenesis is suppressed in mice lacking the metalloproteinase matrilysin"

    Article Title: Intestinal tumorigenesis is suppressed in mice lacking the metalloproteinase matrilysin

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi:

    Mapping and targeted disruption of the matrilysin gene. ( A ) The BSS backcross panel [(C57BL/6JEi × SPRET/Ei)F 1 ) was used to map the matrilysin locus ( Mmp7 ). The segregation patterns of Mmp7 and several linked loci ( D9Bir4 , Ppib-rs1 , and D9Mit160 ) among the backcross offspring are depicted by the columns of boxes, where each column represents the chromosome inherited from the (C57BL/6JEi × SPRET/Ei)F 1 parent. C57BL/6JEi and SPRET/Ei alleles are indicated by the filled and open boxes, respectively. The number at the bottom of each column is the number of offspring with that haplotype. Recombination distances in cM (R) and standard errors (SE) are indicated to the right of the columns. The Chromosome Committee map places the metalloelastase locus ( Mmel ) at the same offset as D9Bir4. D9Bir3 may be the only mapped locus more proximal than Mmp7 . ( B ) Structure of genomic segment, targeting vector, and disrupted allele. The top diagram depicts the 6.5-kb Bam HI (B) genomic fragment containing exons 2–6 (E2-E6; hatched boxes) that was used to generate the targeting construct. Introns, extragenic sequence, and plasmid sequence are denoted by the heavy, thin, and wavy lines, respectively. As shown in the middle diagram, the phosphoglycerate kinase–neomycin cassette containing the phosphoglycerate kinase promoter (filled box), neomycin phosphotransferase cDNA (Neo), and a polyadenylylation signal (+) was inserted using the Eco RV (RV) and Stu I sites (S) indicated. The herpes simplex virus-thymidine kinase (TK) cassette, composed of the TK promoter (barred box) and a polyadenylylation signal (+), was used for negative selection in the presence of gancyclovir. The construct was linearized at the unique Not I (N) site. The bottom diagram shows the expected structure of the targeted allele following electroporation into R1 ES cells and selection for doubly resistant colonies. The locations of the Bam HI and Bst EII (Bs) restriction sites and fragments (probes A and B) used for Southern blotting are also indicated. ( C ) Southern analysis of F 2 progeny from heterozygote matings. Tail DNA was digested with Bst EII and Southern blotted using probe A, which detects bands of 6.3 and 5.3 kb corresponding to the targeted (−) and wild-type (+) alleles, respectively. ( D ) Northern blot analysis of small intestinal RNA. Total RNA was extracted from the entire small intestine of wild-type (+/+), heterozygous (+/−), and homozygous (−/−) F 2 ).
    Figure Legend Snippet: Mapping and targeted disruption of the matrilysin gene. ( A ) The BSS backcross panel [(C57BL/6JEi × SPRET/Ei)F 1 ) was used to map the matrilysin locus ( Mmp7 ). The segregation patterns of Mmp7 and several linked loci ( D9Bir4 , Ppib-rs1 , and D9Mit160 ) among the backcross offspring are depicted by the columns of boxes, where each column represents the chromosome inherited from the (C57BL/6JEi × SPRET/Ei)F 1 parent. C57BL/6JEi and SPRET/Ei alleles are indicated by the filled and open boxes, respectively. The number at the bottom of each column is the number of offspring with that haplotype. Recombination distances in cM (R) and standard errors (SE) are indicated to the right of the columns. The Chromosome Committee map places the metalloelastase locus ( Mmel ) at the same offset as D9Bir4. D9Bir3 may be the only mapped locus more proximal than Mmp7 . ( B ) Structure of genomic segment, targeting vector, and disrupted allele. The top diagram depicts the 6.5-kb Bam HI (B) genomic fragment containing exons 2–6 (E2-E6; hatched boxes) that was used to generate the targeting construct. Introns, extragenic sequence, and plasmid sequence are denoted by the heavy, thin, and wavy lines, respectively. As shown in the middle diagram, the phosphoglycerate kinase–neomycin cassette containing the phosphoglycerate kinase promoter (filled box), neomycin phosphotransferase cDNA (Neo), and a polyadenylylation signal (+) was inserted using the Eco RV (RV) and Stu I sites (S) indicated. The herpes simplex virus-thymidine kinase (TK) cassette, composed of the TK promoter (barred box) and a polyadenylylation signal (+), was used for negative selection in the presence of gancyclovir. The construct was linearized at the unique Not I (N) site. The bottom diagram shows the expected structure of the targeted allele following electroporation into R1 ES cells and selection for doubly resistant colonies. The locations of the Bam HI and Bst EII (Bs) restriction sites and fragments (probes A and B) used for Southern blotting are also indicated. ( C ) Southern analysis of F 2 progeny from heterozygote matings. Tail DNA was digested with Bst EII and Southern blotted using probe A, which detects bands of 6.3 and 5.3 kb corresponding to the targeted (−) and wild-type (+) alleles, respectively. ( D ) Northern blot analysis of small intestinal RNA. Total RNA was extracted from the entire small intestine of wild-type (+/+), heterozygous (+/−), and homozygous (−/−) F 2 ).

    Techniques Used: Plasmid Preparation, Construct, Sequencing, Selection, Electroporation, Southern Blot, Northern Blot

    Localization of matrilysin and gelatinase A in Min /+ tumors. In situ hybridization of matrilysin ( A and B ) and gelatinase A ( C and D ). Photomicrographs were taken using a double exposure with a red filter on dark field illumination so that the silver grains appear pink. ( A ) In a Min colonic adenoma, matrilysin mRNA is expressed by tumor (T) epithelium but not normal (N) colonic mucosa. (×125.) ( B ) Matrilysin mRNA localized to the dysplastic epithelium of a small intestinal tumor (arrow) and is absent in normal intestinal glandular epithelium. (×400.) ( C ) Gelatinase A mRNA is expressed in the tumor (T) compared with the adjacent normal (N) mucosa in a serial section of the colonic tumor shown in A . (×125.) ( D ) Gelatinase A mRNA localizes within the stroma (arrow) of a colonic tumor. (×250.) Immunolocalization of matrilysin protein ( E and F ). Matrilysin protein was visualized with TrueBlue color substrate (dark blue to purple), and nuclei were counterstained with Contrast Red. ( E ) Sporadic localization of matrilysin protein in a small intestinal tumor (T). (×160.) ( F ) Matrilysin immunoreactivity is frequently observed within the lumen of glandular structures (arrow). (×640.)
    Figure Legend Snippet: Localization of matrilysin and gelatinase A in Min /+ tumors. In situ hybridization of matrilysin ( A and B ) and gelatinase A ( C and D ). Photomicrographs were taken using a double exposure with a red filter on dark field illumination so that the silver grains appear pink. ( A ) In a Min colonic adenoma, matrilysin mRNA is expressed by tumor (T) epithelium but not normal (N) colonic mucosa. (×125.) ( B ) Matrilysin mRNA localized to the dysplastic epithelium of a small intestinal tumor (arrow) and is absent in normal intestinal glandular epithelium. (×400.) ( C ) Gelatinase A mRNA is expressed in the tumor (T) compared with the adjacent normal (N) mucosa in a serial section of the colonic tumor shown in A . (×125.) ( D ) Gelatinase A mRNA localizes within the stroma (arrow) of a colonic tumor. (×250.) Immunolocalization of matrilysin protein ( E and F ). Matrilysin protein was visualized with TrueBlue color substrate (dark blue to purple), and nuclei were counterstained with Contrast Red. ( E ) Sporadic localization of matrilysin protein in a small intestinal tumor (T). (×160.) ( F ) Matrilysin immunoreactivity is frequently observed within the lumen of glandular structures (arrow). (×640.)

    Techniques Used: In Situ Hybridization

    36) Product Images from "ClC-2 knockdown prevents cerebrovascular remodeling via inhibition of the Wnt/β-catenin signaling pathway"

    Article Title: ClC-2 knockdown prevents cerebrovascular remodeling via inhibition of the Wnt/β-catenin signaling pathway

    Journal: Cellular & Molecular Biology Letters

    doi: 10.1186/s11658-018-0095-z

    ClC-2 inhibition attenuated the AngII-induced activation of Wnt/β-catenin signaling. a through f HBVSMCs were transfected with ClC-2 siRNA (siClC-2; 20 nM) or negative siRNA (negative; 20 nM) and then stimulated with angiotensin II (AngII; 10 − 7 M) for 48 h. Shown are the western blotting results for β-catenin phosphorylation ( a ), β-catenin cytosol ( b ) and nuclear protein ( c ) levels, GSK-3β phosphorylation ( d ), and survivin ( e ) and cyclin D1 ( f ) protein expression. g Quantitative real-time PCR analysis of Wnt3a and Wnt5a mRNA expression. h The cells were treated with recombinant Wnt3a (100 ng/ml) for 48 h. Wnt3a expression was examined using quantitative real-time. i Viability of HBVSMCs transfected with ClC-2 siRNA followed by co-incubation with recombinant Wnt3a and AngII. ** p
    Figure Legend Snippet: ClC-2 inhibition attenuated the AngII-induced activation of Wnt/β-catenin signaling. a through f HBVSMCs were transfected with ClC-2 siRNA (siClC-2; 20 nM) or negative siRNA (negative; 20 nM) and then stimulated with angiotensin II (AngII; 10 − 7 M) for 48 h. Shown are the western blotting results for β-catenin phosphorylation ( a ), β-catenin cytosol ( b ) and nuclear protein ( c ) levels, GSK-3β phosphorylation ( d ), and survivin ( e ) and cyclin D1 ( f ) protein expression. g Quantitative real-time PCR analysis of Wnt3a and Wnt5a mRNA expression. h The cells were treated with recombinant Wnt3a (100 ng/ml) for 48 h. Wnt3a expression was examined using quantitative real-time. i Viability of HBVSMCs transfected with ClC-2 siRNA followed by co-incubation with recombinant Wnt3a and AngII. ** p

    Techniques Used: Inhibition, Activation Assay, Transfection, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Recombinant, Incubation

    37) Product Images from "Active and water-soluble form of lipidated Wnt protein is maintained by a serum glycoprotein afamin/α-albumin"

    Article Title: Active and water-soluble form of lipidated Wnt protein is maintained by a serum glycoprotein afamin/α-albumin

    Journal: eLife

    doi: 10.7554/eLife.11621

    Identification of the 40-kDa band as Wnt5a. The SEC fractions 1–14 in Figure 6B were subjected to an immunoblotting with anti-mouse Wnt5a monoclonal antibody. The minor band migrating at ~20 kDa likely represents a truncated fragment of Wnt5a. DOI: http://dx.doi.org/10.7554/eLife.11621.016
    Figure Legend Snippet: Identification of the 40-kDa band as Wnt5a. The SEC fractions 1–14 in Figure 6B were subjected to an immunoblotting with anti-mouse Wnt5a monoclonal antibody. The minor band migrating at ~20 kDa likely represents a truncated fragment of Wnt5a. DOI: http://dx.doi.org/10.7554/eLife.11621.016

    Techniques Used: Size-exclusion Chromatography

    Expression/secretion profile of all human Wnt constructs in the presence of serum. N-terminally PA-tagged human Wnt constructs (constructs #4 - #22 shown in Supplementary file 1 ) are singly transfected into HEK293T cells in the presence of 10% bovine serum and the resultant culture supernatants were subjected to the immunoprecipitation with NZ-1 followed by immunoblotting with biotinylated NZ-1 as in Figure 7B . Asterisks denote nonspecific bands present in all samples including that obtained with mock-transfected culture supernatant. DOI: http://dx.doi.org/10.7554/eLife.11621.020
    Figure Legend Snippet: Expression/secretion profile of all human Wnt constructs in the presence of serum. N-terminally PA-tagged human Wnt constructs (constructs #4 - #22 shown in Supplementary file 1 ) are singly transfected into HEK293T cells in the presence of 10% bovine serum and the resultant culture supernatants were subjected to the immunoprecipitation with NZ-1 followed by immunoblotting with biotinylated NZ-1 as in Figure 7B . Asterisks denote nonspecific bands present in all samples including that obtained with mock-transfected culture supernatant. DOI: http://dx.doi.org/10.7554/eLife.11621.020

    Techniques Used: Expressing, Construct, Transfection, Immunoprecipitation

    AFM and Wnt3a form stable 1:1 complex in the absence of detergents. ( A,B ) SEC profiles of affinity-purified Wnt3a preparation in the absence ( A , –CHAPS) or presence ( B , +CHAPS) of 1% CHAPS. Graphs are expanded view of the areas indicated by dotted line box in the whole chromatograms ( insets ). Elusion positions for molecular mass standards including thyroglobulin (669 kDa), ferritin (440 kDa), aldorase (158 kDa) and ovalbumin (44 kDa) are indicated at the top . Sixteen fractions collected from each chromatography are subjected to nonreducing SDS-PAGE on 5–20% gradient gels, followed by Coomassie Blue staining ( C ) or immunoblotting with anti-Wnt3a ( D ), anti-bovine AFM ( E ), or anti-bovine ApoA1 ( F ). In ( C ) ~ ( F ), analysis of the samples from ( A ) and ( B ) are shown in the left (–CHAPS) or right (+CHAPS) panels, respectively. DOI: http://dx.doi.org/10.7554/eLife.11621.008
    Figure Legend Snippet: AFM and Wnt3a form stable 1:1 complex in the absence of detergents. ( A,B ) SEC profiles of affinity-purified Wnt3a preparation in the absence ( A , –CHAPS) or presence ( B , +CHAPS) of 1% CHAPS. Graphs are expanded view of the areas indicated by dotted line box in the whole chromatograms ( insets ). Elusion positions for molecular mass standards including thyroglobulin (669 kDa), ferritin (440 kDa), aldorase (158 kDa) and ovalbumin (44 kDa) are indicated at the top . Sixteen fractions collected from each chromatography are subjected to nonreducing SDS-PAGE on 5–20% gradient gels, followed by Coomassie Blue staining ( C ) or immunoblotting with anti-Wnt3a ( D ), anti-bovine AFM ( E ), or anti-bovine ApoA1 ( F ). In ( C ) ~ ( F ), analysis of the samples from ( A ) and ( B ) are shown in the left (–CHAPS) or right (+CHAPS) panels, respectively. DOI: http://dx.doi.org/10.7554/eLife.11621.008

    Techniques Used: Size-exclusion Chromatography, Affinity Purification, Chromatography, SDS Page, Staining

    38) Product Images from "Dissociated expression of mitochondrial and cytosolic creatine kinases in the human brain: a new perspective on the role of creatine in brain energy metabolism"

    Article Title: Dissociated expression of mitochondrial and cytosolic creatine kinases in the human brain: a new perspective on the role of creatine in brain energy metabolism

    Journal: Journal of Cerebral Blood Flow & Metabolism

    doi: 10.1038/jcbfm.2013.84

    Expression of brain-type creatine kinase (BCK) and ubiquitous mitochondrial creatine kinase (uMtCK) in the cerebellum. ( A , B ) The 3,3′-diaminobenzidine tetrahydrochloride (DAB)–peroxidase staining of ( A ) uMtCK and ( B ) BCK in the cerebellar
    Figure Legend Snippet: Expression of brain-type creatine kinase (BCK) and ubiquitous mitochondrial creatine kinase (uMtCK) in the cerebellum. ( A , B ) The 3,3′-diaminobenzidine tetrahydrochloride (DAB)–peroxidase staining of ( A ) uMtCK and ( B ) BCK in the cerebellar

    Techniques Used: Expressing, Staining

    39) Product Images from "rAAV2/7 vector-mediated overexpression of alpha-synuclein in mouse substantia nigra induces protein aggregation and progressive dose-dependent neurodegeneration"

    Article Title: rAAV2/7 vector-mediated overexpression of alpha-synuclein in mouse substantia nigra induces protein aggregation and progressive dose-dependent neurodegeneration

    Journal: Molecular Neurodegeneration

    doi: 10.1186/1750-1326-8-44

    Soluble and insoluble α-synuclein levels increase over time upon delivery of rAAV2/7-α-synuclein WT or A53T. (A) Western blot and (B) quantitative analysis (n = 3) of soluble α-synuclein in the cytoplasmic (TBS soluble) fraction of SN mouse lysates injected with rAAV2/7-eGFP (4 weeks time point) or rAAV2/7-α-synuclein WT or A53T (5 days, 2 weeks and 4 weeks time points). (C) Immunoblotting and (D) quantification (n = 3) of phosphorylated and detergent-insoluble α-synuclein in the Urea soluble fraction of injected mouse nigral homogenates. For detection, immunoblots for α-synuclein were performed using a panel of different antibodies against: mouse and human α-synuclein (Syn1), specific human α-synuclein (15G7) and P-S129 α-synuclein. GAPDH and β-actin serve as internal loading control in the TBS and Urea fractions, respectively.
    Figure Legend Snippet: Soluble and insoluble α-synuclein levels increase over time upon delivery of rAAV2/7-α-synuclein WT or A53T. (A) Western blot and (B) quantitative analysis (n = 3) of soluble α-synuclein in the cytoplasmic (TBS soluble) fraction of SN mouse lysates injected with rAAV2/7-eGFP (4 weeks time point) or rAAV2/7-α-synuclein WT or A53T (5 days, 2 weeks and 4 weeks time points). (C) Immunoblotting and (D) quantification (n = 3) of phosphorylated and detergent-insoluble α-synuclein in the Urea soluble fraction of injected mouse nigral homogenates. For detection, immunoblots for α-synuclein were performed using a panel of different antibodies against: mouse and human α-synuclein (Syn1), specific human α-synuclein (15G7) and P-S129 α-synuclein. GAPDH and β-actin serve as internal loading control in the TBS and Urea fractions, respectively.

    Techniques Used: Western Blot, Injection

    rAAV2/7 vector-mediated overexpression of α-synuclein in mouse SN induces progressive cell loss and α-synuclein aggregation. (A) Images of immunohistochemical stainings for α-synuclein showing the whole area of transduction 5 days after rAAV2/7-α-synuclein injection. Scale bar = 500 μm. (B) Overexpression of WT α-synuclein induces a progressive and dose-dependent α-synuclein-positive cell loss over time in the injected SN. Absence of immunoreactivity in the contralateral SN at 5 days after injection when expression was maximal. Right panels are magnifications of the overview (left panels). Scale bars = 200 μm. N-inj: non-injected side. (C) High magnification pictures demonstrate the presence of α-synuclein-positive aggregates (arrows) in the SN for each vector dose at 4 weeks post-injection. (D) High magnification picture of an α-synuclein-positive cell without aggregates. Scale bars (C-D) = 5 μm. (E) Stereological quantification of the α-synuclein-positive volume in the SN of mice injected with 3 different WT α-synuclein vector doses and a unique A53T α-synuclein vector dose after 5 days, 4 weeks and 8 weeks. (F) Stereological quantification of the number of α-synuclein-positive cells in the SN of mice after 5 days, 4 weeks and 8 weeks. Asterisks (*) depict significant decrease respective to 4 weeks, unless specified otherwise. 5 days 4,0E + 11 GC/ml WT/A53T: n = 3; 5 days 2,6/8,0E + 11 GC/ml WT: n = 4; 4 weeks: n = 4; 8 weeks: n = 4.
    Figure Legend Snippet: rAAV2/7 vector-mediated overexpression of α-synuclein in mouse SN induces progressive cell loss and α-synuclein aggregation. (A) Images of immunohistochemical stainings for α-synuclein showing the whole area of transduction 5 days after rAAV2/7-α-synuclein injection. Scale bar = 500 μm. (B) Overexpression of WT α-synuclein induces a progressive and dose-dependent α-synuclein-positive cell loss over time in the injected SN. Absence of immunoreactivity in the contralateral SN at 5 days after injection when expression was maximal. Right panels are magnifications of the overview (left panels). Scale bars = 200 μm. N-inj: non-injected side. (C) High magnification pictures demonstrate the presence of α-synuclein-positive aggregates (arrows) in the SN for each vector dose at 4 weeks post-injection. (D) High magnification picture of an α-synuclein-positive cell without aggregates. Scale bars (C-D) = 5 μm. (E) Stereological quantification of the α-synuclein-positive volume in the SN of mice injected with 3 different WT α-synuclein vector doses and a unique A53T α-synuclein vector dose after 5 days, 4 weeks and 8 weeks. (F) Stereological quantification of the number of α-synuclein-positive cells in the SN of mice after 5 days, 4 weeks and 8 weeks. Asterisks (*) depict significant decrease respective to 4 weeks, unless specified otherwise. 5 days 4,0E + 11 GC/ml WT/A53T: n = 3; 5 days 2,6/8,0E + 11 GC/ml WT: n = 4; 4 weeks: n = 4; 8 weeks: n = 4.

    Techniques Used: Plasmid Preparation, Over Expression, Immunohistochemistry, Transduction, Injection, Expressing, Mouse Assay

    α-Synuclein overexpression leads to progressive dose-dependent dopaminergic cell death in mouse SN and STR. (A-B) Immunohistochemical stainings for TH in the (A) SN and in the (B) STR of mice injected with 3 different WT α-synuclein vector doses and a unique eGFP or A53T α-synuclein vector dose show that overexpression of α-synuclein induces a progressive and dose-dependent TH cell loss over time. Overexpression of A53T α-synuclein triggers a comparable loss to WT α-synuclein at the same vector dose. Note the absence of dopaminergic neurodegeneration in the contralateral side at 8 weeks post-injection when cell loss was maximal. Scale bar (A) = 250 μm and (B) = 500 μm. N-inj: non-injected side. (C-D) Stereological quantification of the number of TH-positive neurons in the SN of the (C) injected side and (D) non-injected side after 5 days, 4 weeks and 8 weeks. (E) Stereological quantification of the TH-positive volume in the STR of the injected side at 5 days, 4 weeks and 8 weeks post-injection. Asterisks (*) represent significant decrease respective to eGFP, unless specified otherwise. Dagger (‡) depicts significant decrease respective to 4,0E + 11 GC/ml WT/A53T. 5 days 8,0E + 11 GC/ml eGFP and 4,0E + 11 GC/ml WT/A53T: n = 3; 5 days 2,6/8,0E + 11 GC/ml WT: n = 4; 4 weeks: n = 4; 8 weeks: n = 4.
    Figure Legend Snippet: α-Synuclein overexpression leads to progressive dose-dependent dopaminergic cell death in mouse SN and STR. (A-B) Immunohistochemical stainings for TH in the (A) SN and in the (B) STR of mice injected with 3 different WT α-synuclein vector doses and a unique eGFP or A53T α-synuclein vector dose show that overexpression of α-synuclein induces a progressive and dose-dependent TH cell loss over time. Overexpression of A53T α-synuclein triggers a comparable loss to WT α-synuclein at the same vector dose. Note the absence of dopaminergic neurodegeneration in the contralateral side at 8 weeks post-injection when cell loss was maximal. Scale bar (A) = 250 μm and (B) = 500 μm. N-inj: non-injected side. (C-D) Stereological quantification of the number of TH-positive neurons in the SN of the (C) injected side and (D) non-injected side after 5 days, 4 weeks and 8 weeks. (E) Stereological quantification of the TH-positive volume in the STR of the injected side at 5 days, 4 weeks and 8 weeks post-injection. Asterisks (*) represent significant decrease respective to eGFP, unless specified otherwise. Dagger (‡) depicts significant decrease respective to 4,0E + 11 GC/ml WT/A53T. 5 days 8,0E + 11 GC/ml eGFP and 4,0E + 11 GC/ml WT/A53T: n = 3; 5 days 2,6/8,0E + 11 GC/ml WT: n = 4; 4 weeks: n = 4; 8 weeks: n = 4.

    Techniques Used: Over Expression, Immunohistochemistry, Mouse Assay, Injection, Plasmid Preparation

    Phosphorylation of α-synuclein at S129 increases over time in a dose-dependent manner after rAAV2/7-α-synuclein delivery. (A) Representative images of P-S129 α-synuclein expression in the SN of mice injected with 3 different WT α-synuclein vector doses and a unique A53T α-synuclein vector dose show that overexpression of WT α-synuclein induces a progressive and dose-dependent increase over time in P-S129 α-synuclein. Lack of immunoreactivity in the contralateral SN at 8 weeks after injection when expression was maximal. Right panels are magnifications of the overview (left panels). Scale bars = 200 μm. (B) Stereological quantification of the number of P-S129 α-synuclein positive cells in the injected SN after 5 days, 4 weeks and 8 weeks. (C) Percentage of P-S129 α-synuclein positive cells in the SN at 5 days, 4 weeks and 8 weeks post-injection. Asterisks (*) depict significant increase respective to 5 days, unless specified otherwise. 5 days 4,0E + 11 GC/ml WT/A53T: n = 3; 5 days 2,6/8,0E + 11 GC/ml WT: n = 4; 4 weeks: n = 4; 8 weeks: n = 4.
    Figure Legend Snippet: Phosphorylation of α-synuclein at S129 increases over time in a dose-dependent manner after rAAV2/7-α-synuclein delivery. (A) Representative images of P-S129 α-synuclein expression in the SN of mice injected with 3 different WT α-synuclein vector doses and a unique A53T α-synuclein vector dose show that overexpression of WT α-synuclein induces a progressive and dose-dependent increase over time in P-S129 α-synuclein. Lack of immunoreactivity in the contralateral SN at 8 weeks after injection when expression was maximal. Right panels are magnifications of the overview (left panels). Scale bars = 200 μm. (B) Stereological quantification of the number of P-S129 α-synuclein positive cells in the injected SN after 5 days, 4 weeks and 8 weeks. (C) Percentage of P-S129 α-synuclein positive cells in the SN at 5 days, 4 weeks and 8 weeks post-injection. Asterisks (*) depict significant increase respective to 5 days, unless specified otherwise. 5 days 4,0E + 11 GC/ml WT/A53T: n = 3; 5 days 2,6/8,0E + 11 GC/ml WT: n = 4; 4 weeks: n = 4; 8 weeks: n = 4.

    Techniques Used: Expressing, Mouse Assay, Injection, Plasmid Preparation, Over Expression

    rAAV2/7 vector-mediated α-synuclein overexpression in mouse SN leads to motor behaviour impairments. (A) Performance in the cylinder test of rAAV2/7-eGFP and rAAV2/7-α-synuclein WT mice at 1 week, 4 weeks, 8 weeks and 12 weeks after injection. No asymmetry in forepaw use was detected up to 8 weeks. At 12 weeks after injection, a significantly reduced use of the contralateral forepaw was observed for the mid dose of α-synuclein vector. (B) The rotarod test performed at 14 weeks after injection showed a significant reduction in average time on the rotating rod for both rAAV2/7-α-synuclein doses. (C) A significant decline in the walking distance was observed for the two α-synuclein doses tested in the open field at 15 weeks after injection. For all tests: 8,0E + 11 GC/ml eGFP/WT and 4,0E + 11 GC/ml WT: n = 10.
    Figure Legend Snippet: rAAV2/7 vector-mediated α-synuclein overexpression in mouse SN leads to motor behaviour impairments. (A) Performance in the cylinder test of rAAV2/7-eGFP and rAAV2/7-α-synuclein WT mice at 1 week, 4 weeks, 8 weeks and 12 weeks after injection. No asymmetry in forepaw use was detected up to 8 weeks. At 12 weeks after injection, a significantly reduced use of the contralateral forepaw was observed for the mid dose of α-synuclein vector. (B) The rotarod test performed at 14 weeks after injection showed a significant reduction in average time on the rotating rod for both rAAV2/7-α-synuclein doses. (C) A significant decline in the walking distance was observed for the two α-synuclein doses tested in the open field at 15 weeks after injection. For all tests: 8,0E + 11 GC/ml eGFP/WT and 4,0E + 11 GC/ml WT: n = 10.

    Techniques Used: Plasmid Preparation, Over Expression, Mouse Assay, Injection

    Efficient dopaminergic neuron transduction upon rAAV2/7 vector delivery in mouse SN. (A-B) Representative confocal images of fluorescent double immunostainings for α-synuclein (green) and TH (red) at (A) 5 days and at (B) 8 weeks after injection of rAAV2/7-α-synuclein WT at 8,0E + 11 GC/ml. (A) At 5 days post-injection, pictures reveal extensive co-localization (merge) in the transduced region. Bottom panels are magnifications of the overviews (upper panels). Scale bar upper panel = 200 μm and bottom panel = 50 μm. Confocal images of double immunostainings for α-synuclein (green) and NeuN or GFAP (red) show an almost exclusive neuronal transduction. Scale bar = 100 μm. (B) At 8 weeks after injection, a clear degeneration of the nigral dopaminergic neurons is observed upon rAAV2/7 vector-mediated α-synuclein overexpression. Dopaminergic neurons remain widely present in the contralateral side. (C-D) Fluorescent double stainings for GFP (green) and TH (red) at (C) 5 days and at (D) 8 weeks post-injection show that rAAV2/7-eGFP at 8,0E + 11 GC/ml does not induce any dopaminergic cell death over time. Inj: injected side; N-inj: non-injected side.
    Figure Legend Snippet: Efficient dopaminergic neuron transduction upon rAAV2/7 vector delivery in mouse SN. (A-B) Representative confocal images of fluorescent double immunostainings for α-synuclein (green) and TH (red) at (A) 5 days and at (B) 8 weeks after injection of rAAV2/7-α-synuclein WT at 8,0E + 11 GC/ml. (A) At 5 days post-injection, pictures reveal extensive co-localization (merge) in the transduced region. Bottom panels are magnifications of the overviews (upper panels). Scale bar upper panel = 200 μm and bottom panel = 50 μm. Confocal images of double immunostainings for α-synuclein (green) and NeuN or GFAP (red) show an almost exclusive neuronal transduction. Scale bar = 100 μm. (B) At 8 weeks after injection, a clear degeneration of the nigral dopaminergic neurons is observed upon rAAV2/7 vector-mediated α-synuclein overexpression. Dopaminergic neurons remain widely present in the contralateral side. (C-D) Fluorescent double stainings for GFP (green) and TH (red) at (C) 5 days and at (D) 8 weeks post-injection show that rAAV2/7-eGFP at 8,0E + 11 GC/ml does not induce any dopaminergic cell death over time. Inj: injected side; N-inj: non-injected side.

    Techniques Used: Transduction, Plasmid Preparation, Injection, Over Expression

    40) Product Images from "Somatodendritic Depolarization-Activated Potassium Currents in Rat Neostriatal Cholinergic Interneurons Are Predominantly of the A Type and Attributable to Coexpression of Kv4.2 and Kv4.1 Subunits"

    Article Title: Somatodendritic Depolarization-Activated Potassium Currents in Rat Neostriatal Cholinergic Interneurons Are Predominantly of the A Type and Attributable to Coexpression of Kv4.2 and Kv4.1 Subunits

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.18-09-03124.1998

    The somatodendritic membrane of cholinergic interneurons possesses Kv4.2 but not Kv1.4 immunoreactivity. A , Immunohistochemical localization of Kv1.4 in the striatum. Kv1.4-immunoreactive fiber bundles ( arrowheads ) course through the striatum to the globus pallidus ( GP ). Neuronal somata within the striatum show no immunoreactivity to Kv1.4. Note a dense Kv1.4 staining in the GP. B , Immunohistochemical localization of Kv4.2C in the striatum. Medium to large somata ( arrowheads ) were densely labeled with Kv4.2C, whereas small cells were weakly labeled. Note that proximal dendrites of medium to large cells were also labeled. C, D , Examples of striatal neurons double-labeled for Kv4.2C ( C ) and ChAT ( D ). C , Two neurons labeled for Kv4.2C with FITC. D , the same two neurons were labeled for ChAT and visualized with Texas Red. Scale bars: A , 50 μm; B , 100 μm; C, D , 50 μm.
    Figure Legend Snippet: The somatodendritic membrane of cholinergic interneurons possesses Kv4.2 but not Kv1.4 immunoreactivity. A , Immunohistochemical localization of Kv1.4 in the striatum. Kv1.4-immunoreactive fiber bundles ( arrowheads ) course through the striatum to the globus pallidus ( GP ). Neuronal somata within the striatum show no immunoreactivity to Kv1.4. Note a dense Kv1.4 staining in the GP. B , Immunohistochemical localization of Kv4.2C in the striatum. Medium to large somata ( arrowheads ) were densely labeled with Kv4.2C, whereas small cells were weakly labeled. Note that proximal dendrites of medium to large cells were also labeled. C, D , Examples of striatal neurons double-labeled for Kv4.2C ( C ) and ChAT ( D ). C , Two neurons labeled for Kv4.2C with FITC. D , the same two neurons were labeled for ChAT and visualized with Texas Red. Scale bars: A , 50 μm; B , 100 μm; C, D , 50 μm.

    Techniques Used: Immunohistochemistry, Staining, Labeling

    Related Articles

    Staining:

    Article Title: In Utero Exposure to Valproic Acid Induces Neocortical Dysgenesis via Dysregulation of Neural Progenitor Cell Proliferation/Differentiation
    Article Snippet: .. The sections were immunohistochemically stained using anti-GABA antibody (1:1000; A2052, Sigma-Aldrich, RRID: AB_477652; 4°C, overnight) as a primary antibody, Alexa Fluor 555 anti-rabbit IgG antibody (1:600; A-31572, Thermo Fischer Scientific, RRID: AB_10562716; room temperature, 1.5 h) as a secondary antibody, and bisbenzimide trihydrochloride (1:300 of 1% solution; , Sigma-Aldrich; room temperature, 1.5 h) as a counterstaining reagent. ..

    Immunohistochemistry:

    Article Title: Assembly of Functional Forebrain Spheroids from Human Pluripotent Cells
    Article Snippet: .. The following primary antibodies were used for immunohistochemistry: anti–NKX2.1 (rabbit, 1:200; Santa Cruz: sc-13040), anti–MAP2 (guinea pig, 1:1,000; Synaptic Systems: 188004), anti–GABA (rabbit, 1:1,000; Sigma: A2052), anti–GAD67 (mouse, 1:1,000; Millipore: MAB5406), anti–SST (rat, 1:200; Millipore: MAB354), anti–CR (rabbit, 1:1,000; Swant: CR7697), anti–CB (rabbit, 1:1,000; Swant: CB38), anti–PV (rabbit, 1:6,000; Swant: PV27), anti–PV (mouse 1:1,000; Millipore: MAB1572), anti–GFP (chicken, 1:1,500; GeneTex: GTX13970), anti–DCX (guinea pig, 1:1,000; Millipore: AB2253); anti–TBR1 (rabbit, 1:200; Abcam: AB31940), anti–GFAP (rabbit, 1:1,000; DAKO Z0334), anti–CTIP2 (rat, 1:300; Abcam: AB18465), anti–OCT4 (rabbit, 1:200, Cell Signaling Technology), anti–SSEA4 (mouse, 1:200, Cell Signaling Technology). .. AlexaFluo Dyes (Life Technologies) were used at 1:1000 dilution for amplifying the signal.

    Affinity Purification:

    Article Title: Cre-Mediated Recombination in Rhombic Lip Derivatives
    Article Snippet: .. Immunofluorescence was performed as described ( ) on free-floating 40 μm sections using monoclonal anti-β-galactosidase (Promega 1:500), rabbit anti- GABA (Sigma A-2052 1:2000), affinity-purified rabbit anti- GABAA receptor α6 subunit (generously provided by F.A. ..

    Immunofluorescence:

    Article Title: Cre-Mediated Recombination in Rhombic Lip Derivatives
    Article Snippet: .. Immunofluorescence was performed as described ( ) on free-floating 40 μm sections using monoclonal anti-β-galactosidase (Promega 1:500), rabbit anti- GABA (Sigma A-2052 1:2000), affinity-purified rabbit anti- GABAA receptor α6 subunit (generously provided by F.A. ..

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