biotinylated anti rabbit antibody  (Vector Laboratories)


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    Name:
    Biotinylated Goat Anti Rabbit IgG Antibody
    Description:
    Biotinylated Goat Anit Rabbit IgG Antibody is prepared using proprietary immunization schedules that produce high affinity antibodies The antibodies are then purified by affinity chromatography and cross reactivities that are likely to interfere with specific labeling are removed by solid phase adsorption techniques The biotinylated secondary antibodies are conjugated to ensure the maximum degree of labeling without compromising the specificity or affinity of the antibody These antibodies are subjected to rigorous quality control assays and can be used for tissue and cell staining ELISAs and blots Biotinylated Goat Anti Rabbit IgG H L is supplied in liquid format With some exceptions the recommended dilution for most applications is 1 200 H L indicates the antibody recognizes both heavy and light chains This antibody is included in the VECTASTAIN ABC kits
    Catalog Number:
    ba-1000
    Price:
    None
    Category:
    Antibodies
    Reactivity:
    Rabbit
    Size:
    1 5 mg
    Host:
    Goat
    Buy from Supplier


    Structured Review

    Vector Laboratories biotinylated anti rabbit antibody
    Biotinylated Goat Anti Rabbit IgG Antibody
    Biotinylated Goat Anit Rabbit IgG Antibody is prepared using proprietary immunization schedules that produce high affinity antibodies The antibodies are then purified by affinity chromatography and cross reactivities that are likely to interfere with specific labeling are removed by solid phase adsorption techniques The biotinylated secondary antibodies are conjugated to ensure the maximum degree of labeling without compromising the specificity or affinity of the antibody These antibodies are subjected to rigorous quality control assays and can be used for tissue and cell staining ELISAs and blots Biotinylated Goat Anti Rabbit IgG H L is supplied in liquid format With some exceptions the recommended dilution for most applications is 1 200 H L indicates the antibody recognizes both heavy and light chains This antibody is included in the VECTASTAIN ABC kits
    https://www.bioz.com/result/biotinylated anti rabbit antibody/product/Vector Laboratories
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    biotinylated anti rabbit antibody - by Bioz Stars, 2021-03
    97/100 stars

    Images

    1) Product Images from "Fluorescent Arc/Arg3.1 indicator mice: a versatile tool to study brain activity changes in vitro and in vivo"

    Article Title: Fluorescent Arc/Arg3.1 indicator mice: a versatile tool to study brain activity changes in vitro and in vivo

    Journal: Journal of neuroscience methods

    doi: 10.1016/j.jneumeth.2009.07.015

    Increased d4EGFP expression in a mouse model of Alzheimer’s disease Representative images of DG hippocampus (coronal sections) from one month-old Tg Arc/Arg3.1-d4EGFP and Tg 5XFAD /Tg Arc/Arg3.1-d4EGFP littermate mice are shown after visualization of d4EGFP by DAB (A, B) or immunofluorescence (C,D). Note the increased number of and labeling intensity of d4EGFP-positive neurons in CA1 (B) and DG granule cells (B, D) in the Tg 5XFAD /Tg Arc/Arg3.1-d4EGFP mice. Brain sections stained with anti-GFP antibody and visualized by biotinylated (DAB method) or FITC-conjugated secondary antibodies. The fluorescent images are single confocal scans. Abbreviations: db – dorsal blade of DG, vb – ventral blade of DG; scale bars = 250 μm (A,B) and 100 μm (C,D). (E) Count of d4EGFP-positive neurons in Tg 5XFAD /Tg Arc/Arg3.1-d4EGFP mice, as percent change from control Tg Arc/Arg3.1-d4EGFP littermates, in the DG: (% mean ± SD): 216.3 ± 7.7 vs. 100 ± 6.4 (**p
    Figure Legend Snippet: Increased d4EGFP expression in a mouse model of Alzheimer’s disease Representative images of DG hippocampus (coronal sections) from one month-old Tg Arc/Arg3.1-d4EGFP and Tg 5XFAD /Tg Arc/Arg3.1-d4EGFP littermate mice are shown after visualization of d4EGFP by DAB (A, B) or immunofluorescence (C,D). Note the increased number of and labeling intensity of d4EGFP-positive neurons in CA1 (B) and DG granule cells (B, D) in the Tg 5XFAD /Tg Arc/Arg3.1-d4EGFP mice. Brain sections stained with anti-GFP antibody and visualized by biotinylated (DAB method) or FITC-conjugated secondary antibodies. The fluorescent images are single confocal scans. Abbreviations: db – dorsal blade of DG, vb – ventral blade of DG; scale bars = 250 μm (A,B) and 100 μm (C,D). (E) Count of d4EGFP-positive neurons in Tg 5XFAD /Tg Arc/Arg3.1-d4EGFP mice, as percent change from control Tg Arc/Arg3.1-d4EGFP littermates, in the DG: (% mean ± SD): 216.3 ± 7.7 vs. 100 ± 6.4 (**p

    Techniques Used: Expressing, Mouse Assay, Immunofluorescence, Labeling, Staining

    2) Product Images from "Vasohibin as an endothelium-derived negative feedback regulator of angiogenesis"

    Article Title: Vasohibin as an endothelium-derived negative feedback regulator of angiogenesis

    Journal: Journal of Clinical Investigation

    doi: 10.1172/JCI200421152

    KIAA1036 is preferentially expressed in ECs. ( A ) Expression of KIAA1036 in cultured cells. Cells were preincubated in 0.1% FCS/α-MEM for 12 hours and then stimulated with growth factors as follows: HUVECs with VEGF (1 nM), HASMCs with PDGF (1 nM), human fibroblasts with FGF-2 (2 nM), and keratinocytes with EGF (1 nM). Thereafter, total RNA was obtained and Northern blotting for vasohibin was performed. ( B ) Expression of KIAA1036 in vivo was examined by multiple-tissue Northern blot. ( C ) Localization of KIAA1036 protein in the placenta. Sections of human placenta were subjected to immunostaining. Anti_human CD31 mAb, anti_KIAA1036 mAb, or mouse IgG was used as the primary Ab. Scale bars: 100 μm. ( D ) Expression of KIAA1036 in human embryo. Northern blotting for vasohibin was performed using a human developmental total RNA Northern blot.
    Figure Legend Snippet: KIAA1036 is preferentially expressed in ECs. ( A ) Expression of KIAA1036 in cultured cells. Cells were preincubated in 0.1% FCS/α-MEM for 12 hours and then stimulated with growth factors as follows: HUVECs with VEGF (1 nM), HASMCs with PDGF (1 nM), human fibroblasts with FGF-2 (2 nM), and keratinocytes with EGF (1 nM). Thereafter, total RNA was obtained and Northern blotting for vasohibin was performed. ( B ) Expression of KIAA1036 in vivo was examined by multiple-tissue Northern blot. ( C ) Localization of KIAA1036 protein in the placenta. Sections of human placenta were subjected to immunostaining. Anti_human CD31 mAb, anti_KIAA1036 mAb, or mouse IgG was used as the primary Ab. Scale bars: 100 μm. ( D ) Expression of KIAA1036 in human embryo. Northern blotting for vasohibin was performed using a human developmental total RNA Northern blot.

    Techniques Used: Expressing, Cell Culture, Northern Blot, In Vivo, Immunostaining

    3) Product Images from "Enhanced biocompatibility of CD47-functionalized vascular stents"

    Article Title: Enhanced biocompatibility of CD47-functionalized vascular stents

    Journal: Biomaterials

    doi: 10.1016/j.biomaterials.2016.02.008

    CD47 Quantification. (A) The modification chemistry summarized in was utilized to facilitate quantification. Following PEI-PDT addition, primary amines were acetylated using sulfo-NHS-Acetate. recCD47 and pepCD47 were biotinylated using Sulfo-NHS-LC-LC-Biotin.
    Figure Legend Snippet: CD47 Quantification. (A) The modification chemistry summarized in was utilized to facilitate quantification. Following PEI-PDT addition, primary amines were acetylated using sulfo-NHS-Acetate. recCD47 and pepCD47 were biotinylated using Sulfo-NHS-LC-LC-Biotin.

    Techniques Used: Modification

    4) Product Images from "Combinational therapy using hypothermia and the immunophilin ligand FK506 to target altered pial arteriolar reactivity, axonal damage, and blood-brain barrier dysfunction after traumatic brain injury in rat"

    Article Title: Combinational therapy using hypothermia and the immunophilin ligand FK506 to target altered pial arteriolar reactivity, axonal damage, and blood-brain barrier dysfunction after traumatic brain injury in rat

    Journal: Journal of Cerebral Blood Flow & Metabolism

    doi: 10.1038/jcbfm.2010.208

    Bar graph shows a comparison of the mean area of IgG immunostaining among groups. It must be noted that neither hypothermic intervention nor FK506 administration prevented the spatial extravasation of endogenous IgG. In addition, even the combination
    Figure Legend Snippet: Bar graph shows a comparison of the mean area of IgG immunostaining among groups. It must be noted that neither hypothermic intervention nor FK506 administration prevented the spatial extravasation of endogenous IgG. In addition, even the combination

    Techniques Used: Immunostaining

    5) Product Images from "Insertion of Basic Amino Acids in the Hemagglutinin Cleavage Site of H4N2 Avian Influenza Virus (AIV)—Reduced Virus Fitness in Chickens is Restored by Reassortment with Highly Pathogenic H5N1 AIV"

    Article Title: Insertion of Basic Amino Acids in the Hemagglutinin Cleavage Site of H4N2 Avian Influenza Virus (AIV)—Reduced Virus Fitness in Chickens is Restored by Reassortment with Highly Pathogenic H5N1 AIV

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms21072353

    Distribution of avian influenza virus NP in selected organs of inoculated birds. Distribution of influenza NP in brain ( A , E , I , M ), lung ( B , F , J , N ), spleen ( C , G , K , O ) and heart ( D , H , L , P ) of inoculated chickens of selected viruses at 4 dpi (except for H5N1_H4_T 327 K) as detected by immunohistochemistry using primary polyclonal rabbit anti-NP A/FPV/Rostock/34 antibody (1:750) and a secondary biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA, USA) antibody (1:200). 3-amino-9-ethyl-carbazol (red-brown); hematoxylin counterstain (blue); Nomarski contrast; bars A,B,D,E,F,H,I,J,L,M,N,P = 20 µm. Bars C,G,K,O = 50 µm.
    Figure Legend Snippet: Distribution of avian influenza virus NP in selected organs of inoculated birds. Distribution of influenza NP in brain ( A , E , I , M ), lung ( B , F , J , N ), spleen ( C , G , K , O ) and heart ( D , H , L , P ) of inoculated chickens of selected viruses at 4 dpi (except for H5N1_H4_T 327 K) as detected by immunohistochemistry using primary polyclonal rabbit anti-NP A/FPV/Rostock/34 antibody (1:750) and a secondary biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA, USA) antibody (1:200). 3-amino-9-ethyl-carbazol (red-brown); hematoxylin counterstain (blue); Nomarski contrast; bars A,B,D,E,F,H,I,J,L,M,N,P = 20 µm. Bars C,G,K,O = 50 µm.

    Techniques Used: Immunohistochemistry, Plasmid Preparation

    6) Product Images from "Antibodies to CD20 and MHC class II antigen bound to B-lymphoma cells accumulate in shed cytoplasmic fragments"

    Article Title: Antibodies to CD20 and MHC class II antigen bound to B-lymphoma cells accumulate in shed cytoplasmic fragments

    Journal: British Journal of Cancer

    doi: 10.1038/sj.bjc.6602131

    Immunoperoxidase staining of B-lymphoma cells after overnight incubation with anti-CD20 (1F5). Cells were deposited on cytocentrifuge slides, fixed with formaldehyde, permeabilised with saponin, and stained with a biotinylated horse anti-mouse IgG, followed by a complex of streptavidin and peroxidase. Observation was with an × 40 objective, except as noted. ( A ) Raji cells, showing prominent staining of JN spots. ( B ) RL cells, showing dark staining of apparently extracellular objects, referred to as CFs. ( C ) A lower-power photograph of RL cells (× 10), to show the general staining pattern. ( D ) RL cells stained in the absence of saponin, to show antigen that is accessible without permeabilisation. Control Abs of the same subclass produced no brown staining.
    Figure Legend Snippet: Immunoperoxidase staining of B-lymphoma cells after overnight incubation with anti-CD20 (1F5). Cells were deposited on cytocentrifuge slides, fixed with formaldehyde, permeabilised with saponin, and stained with a biotinylated horse anti-mouse IgG, followed by a complex of streptavidin and peroxidase. Observation was with an × 40 objective, except as noted. ( A ) Raji cells, showing prominent staining of JN spots. ( B ) RL cells, showing dark staining of apparently extracellular objects, referred to as CFs. ( C ) A lower-power photograph of RL cells (× 10), to show the general staining pattern. ( D ) RL cells stained in the absence of saponin, to show antigen that is accessible without permeabilisation. Control Abs of the same subclass produced no brown staining.

    Techniques Used: Immunoperoxidase Staining, Incubation, Staining, Produced

    7) Product Images from "Modulation of Sodium/Iodide Symporter Expression in the Salivary Gland"

    Article Title: Modulation of Sodium/Iodide Symporter Expression in the Salivary Gland

    Journal: Thyroid

    doi: 10.1089/thy.2012.0571

    Sodium/iodide symporter (NIS) expression is primarily restricted to striated ducts in human salivary glands. (A) NIS is expressed at a high level along basolateral membranes of striated duct cells (S), with fewer intercalated (rectangle) and excretory (E) duct cells demonstrating lower NIS levels. Acinar (A) cells do not express NIS. Anti-human NIS antibody, avidin-biotin complex, DAB chromogen, hematoxylin counterstain, bar=25 μm. (B) Distribution and intensity of NIS immunoreactivity among intercalated (ID), striated (SD), and excretory (ED) ductal cells in normal human submandibular, parotid, and minor salivary glands. NIS expression is greater and more frequent in striated ducts, regardless of gland type.
    Figure Legend Snippet: Sodium/iodide symporter (NIS) expression is primarily restricted to striated ducts in human salivary glands. (A) NIS is expressed at a high level along basolateral membranes of striated duct cells (S), with fewer intercalated (rectangle) and excretory (E) duct cells demonstrating lower NIS levels. Acinar (A) cells do not express NIS. Anti-human NIS antibody, avidin-biotin complex, DAB chromogen, hematoxylin counterstain, bar=25 μm. (B) Distribution and intensity of NIS immunoreactivity among intercalated (ID), striated (SD), and excretory (ED) ductal cells in normal human submandibular, parotid, and minor salivary glands. NIS expression is greater and more frequent in striated ducts, regardless of gland type.

    Techniques Used: Expressing, Avidin-Biotin Assay

    NIS expression is reduced in inflamed and neoplastic human salivary glands. (A) NIS expression in striated ducts is decreased in inflammatory salivary glands, as well as in benign and malignant neoplasms of ductal origin. (B) NIS immunoreactivity in inflamed (i, ii) and neoplastic (iii, iv) salivary tissue. NIS expression is variably decreased in striated ducts (S) of chronically inflamed salivary glands (i, ii) , particularly in ducts demonstrating goblet metaplasia (arrows). Neoplastic cells of Warthin's tumor (iii) and mucoepidermoid carcinoma (iv) demonstrate lower NIS level, with immunostaining intensity of 2+ and 1+, respectively. Anti-human NIS antibody, avidin-biotin complex, DAB chromogen, hematoxylin counterstain, bars=25 μm. F, periductal fibrosis; L, lymphocytic inflammation.
    Figure Legend Snippet: NIS expression is reduced in inflamed and neoplastic human salivary glands. (A) NIS expression in striated ducts is decreased in inflammatory salivary glands, as well as in benign and malignant neoplasms of ductal origin. (B) NIS immunoreactivity in inflamed (i, ii) and neoplastic (iii, iv) salivary tissue. NIS expression is variably decreased in striated ducts (S) of chronically inflamed salivary glands (i, ii) , particularly in ducts demonstrating goblet metaplasia (arrows). Neoplastic cells of Warthin's tumor (iii) and mucoepidermoid carcinoma (iv) demonstrate lower NIS level, with immunostaining intensity of 2+ and 1+, respectively. Anti-human NIS antibody, avidin-biotin complex, DAB chromogen, hematoxylin counterstain, bars=25 μm. F, periductal fibrosis; L, lymphocytic inflammation.

    Techniques Used: Expressing, Immunostaining, Avidin-Biotin Assay

    8) Product Images from "Human APOE genotype affects intraneuronal Aβ1–42 accumulation in a lentiviral gene transfer model"

    Article Title: Human APOE genotype affects intraneuronal Aβ1–42 accumulation in a lentiviral gene transfer model

    Journal: Human Molecular Genetics

    doi: 10.1093/hmg/ddt525

    Intracellular accumulation of lentiviral Aβ 1–42 is greatest in APOE4-TR mice at 2 weeks after lentivirus injection. ( A ) Representative image of a coronal brain section with DAB staining of biotinylated MOAB2 for lentiviral Aβ 1–42
    Figure Legend Snippet: Intracellular accumulation of lentiviral Aβ 1–42 is greatest in APOE4-TR mice at 2 weeks after lentivirus injection. ( A ) Representative image of a coronal brain section with DAB staining of biotinylated MOAB2 for lentiviral Aβ 1–42

    Techniques Used: Mouse Assay, Injection, Staining

    9) Product Images from "Differential Caveolin-1 Polarization in Endothelial Cells during Migration in Two and Three Dimensions"

    Article Title: Differential Caveolin-1 Polarization in Endothelial Cells during Migration in Two and Three Dimensions

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E02-11-0761

    Polarization of caveolin-1 in planar-migrating ECs requires neither Tyr 14 phosphorylation nor palmitoylation. (A–C) ECs were transfected with vector encoding GFP and subjected to planar, wound-induced migration for 24 h in the presence of 10 ng ml – 1 basic FGF. The cells were fixed, and GFP was visualized by confocal fluorescence microscopy (A). Cellular caveolin-1 was detected with rabbit anti-caveolin-1 as primary antibody followed by biotinylated goat anti-rabbit antibody and Texas Redavidin (B). Overlay of GFP fluorescence and cellular caveolin-1 immunostaining is shown in the merged image (C). (D–F) ECs were transfected with vector encoding wild-type caveolin-1-GFP, and migrating cells were treated and analyzed as in A–C. (G–I) ECs were transfected with vector expressing GFP-tagged caveolin-1 containing Cys-to-Ser mutations in all three palmitoylation sites; migrating cells were treated and analyzed as in A–C. (J–L) ECs were transfected with vector expressing GFP-tagged caveolin-1 containing an Ala substitution at Tyr 14 ; migrating cells were treated and analyzed as in A–C. (M–O) ECs were transfected with vector expressing GFP-tagged caveolin-1-β; migrating cells were treated and analyzed as in A–C.
    Figure Legend Snippet: Polarization of caveolin-1 in planar-migrating ECs requires neither Tyr 14 phosphorylation nor palmitoylation. (A–C) ECs were transfected with vector encoding GFP and subjected to planar, wound-induced migration for 24 h in the presence of 10 ng ml – 1 basic FGF. The cells were fixed, and GFP was visualized by confocal fluorescence microscopy (A). Cellular caveolin-1 was detected with rabbit anti-caveolin-1 as primary antibody followed by biotinylated goat anti-rabbit antibody and Texas Redavidin (B). Overlay of GFP fluorescence and cellular caveolin-1 immunostaining is shown in the merged image (C). (D–F) ECs were transfected with vector encoding wild-type caveolin-1-GFP, and migrating cells were treated and analyzed as in A–C. (G–I) ECs were transfected with vector expressing GFP-tagged caveolin-1 containing Cys-to-Ser mutations in all three palmitoylation sites; migrating cells were treated and analyzed as in A–C. (J–L) ECs were transfected with vector expressing GFP-tagged caveolin-1 containing an Ala substitution at Tyr 14 ; migrating cells were treated and analyzed as in A–C. (M–O) ECs were transfected with vector expressing GFP-tagged caveolin-1-β; migrating cells were treated and analyzed as in A–C.

    Techniques Used: Transfection, Plasmid Preparation, Migration, Fluorescence, Microscopy, Immunostaining, Expressing

    Polarization of caveolin-1 in transmigrating ECs requires Tyr 14 phosphorylation but not palmitoylation. (A) ECs were transiently transfected with plasmids encoding C-terminally GFP-tagged wild-type caveolin (Cav)-1 (Wt), C-terminally GFP-tagged caveolin-1 mutated at all three palmitoylation sites (Palm mut), or with the vector (Vect) alone. The cells were subjected to metabolic labeling by incubation with [ 3 H]palmitate for 4 h, and caveolin-1 immunoprecipitated (IP) with rabbit anti-caveolin antibody (ab), or with nonimmune, purified rabbit IgG (IgG) as control. The immunoprecipitate was subjected to SDS-PAGE and fluorography. Radiolabeling of plasmid-driven, Cav-1-GFP was detected by film exposure for 6 mo. (B) [ 3 H]Palmitate labeling of endogenous caveolin-1α and -1β in the same gel was detected by film exposure for 1 mo. (C) ECs were transiently transfected with plasmids encoding C-terminally GFP-tagged caveolin-1 with an Ala substitution at Tyr 14 (Tyr mut), with wild-type caveolin-1-GFP, with the palmitoylation mutant form of caveolin-1-GFP, or with the vector alone. After recovery, the cells were incubated with medium or with H 2 O 2 (5 mM) for 20 min. Caveolin-1 was immunoprecipitated and subjected to SDS-PAGE. Phosphocaveolin-1-GFP was detected by immmunoblot analysis with anti-phosphocaveolin-1 antibody. (D) The amount of caveolin-1-GFP expressed by all transfected cells was shown to be nearly identical by reprobing the immmunoblot shown in C with anti-GFP antibody. (E–G) ECs were transfected with vector encoding GFP and permitted to transmigrate for 2 h across a collagen-coated polycarbonate filter toward 10 ng ml – 1 basic FGF. The cells were fixed and GFP was visualized by laser-scanning confocal fluorescence analysis (E). The upper face of the filter is indicated as U. Cellular caveolin-1 was detected with rabbit anticaveolin-1 as primary antibody followed by biotinylated goat anti-rabbit antibody and Texas Red-avidin (F). Overlay of GFP fluorescence and cellular caveolin-1 immunostaining is shown in the merged image (G). (H–J) ECs were transfected with vector encoding wild-type caveolin-1-GFP, and migrating cells were treated and analyzed as in E–G. (K–M) ECs were transfected with vector encoding GFP-tagged caveolin-1 containing Cys-to-Ser mutations in all three palmitoylation sites; migrating cells were treated and analyzed as in E–G. (N–P) ECs were transfected with vector encoding GFP-tagged caveolin-1 containing an Ala substitution at Tyr 14 ; migrating cells were treated and analyzed as in E–G. (Q–S) ECs were transfected with vector encoding GFP-tagged caveolin-1β; migrating cells were treated and analyzed as in E–G.
    Figure Legend Snippet: Polarization of caveolin-1 in transmigrating ECs requires Tyr 14 phosphorylation but not palmitoylation. (A) ECs were transiently transfected with plasmids encoding C-terminally GFP-tagged wild-type caveolin (Cav)-1 (Wt), C-terminally GFP-tagged caveolin-1 mutated at all three palmitoylation sites (Palm mut), or with the vector (Vect) alone. The cells were subjected to metabolic labeling by incubation with [ 3 H]palmitate for 4 h, and caveolin-1 immunoprecipitated (IP) with rabbit anti-caveolin antibody (ab), or with nonimmune, purified rabbit IgG (IgG) as control. The immunoprecipitate was subjected to SDS-PAGE and fluorography. Radiolabeling of plasmid-driven, Cav-1-GFP was detected by film exposure for 6 mo. (B) [ 3 H]Palmitate labeling of endogenous caveolin-1α and -1β in the same gel was detected by film exposure for 1 mo. (C) ECs were transiently transfected with plasmids encoding C-terminally GFP-tagged caveolin-1 with an Ala substitution at Tyr 14 (Tyr mut), with wild-type caveolin-1-GFP, with the palmitoylation mutant form of caveolin-1-GFP, or with the vector alone. After recovery, the cells were incubated with medium or with H 2 O 2 (5 mM) for 20 min. Caveolin-1 was immunoprecipitated and subjected to SDS-PAGE. Phosphocaveolin-1-GFP was detected by immmunoblot analysis with anti-phosphocaveolin-1 antibody. (D) The amount of caveolin-1-GFP expressed by all transfected cells was shown to be nearly identical by reprobing the immmunoblot shown in C with anti-GFP antibody. (E–G) ECs were transfected with vector encoding GFP and permitted to transmigrate for 2 h across a collagen-coated polycarbonate filter toward 10 ng ml – 1 basic FGF. The cells were fixed and GFP was visualized by laser-scanning confocal fluorescence analysis (E). The upper face of the filter is indicated as U. Cellular caveolin-1 was detected with rabbit anticaveolin-1 as primary antibody followed by biotinylated goat anti-rabbit antibody and Texas Red-avidin (F). Overlay of GFP fluorescence and cellular caveolin-1 immunostaining is shown in the merged image (G). (H–J) ECs were transfected with vector encoding wild-type caveolin-1-GFP, and migrating cells were treated and analyzed as in E–G. (K–M) ECs were transfected with vector encoding GFP-tagged caveolin-1 containing Cys-to-Ser mutations in all three palmitoylation sites; migrating cells were treated and analyzed as in E–G. (N–P) ECs were transfected with vector encoding GFP-tagged caveolin-1 containing an Ala substitution at Tyr 14 ; migrating cells were treated and analyzed as in E–G. (Q–S) ECs were transfected with vector encoding GFP-tagged caveolin-1β; migrating cells were treated and analyzed as in E–G.

    Techniques Used: Transfection, Plasmid Preparation, Labeling, Incubation, Immunoprecipitation, Purification, SDS Page, Radioactivity, Mutagenesis, Fluorescence, Avidin-Biotin Assay, Immunostaining

    Caveolin-1 accumulates in the forward extension of transmigrating ECs. ECs were allowed to migrate for 2 h across a collagen-coated polycarbonate filter (8-μm pore size) toward 10 ng ml – 1 basic FGF in the lower compartment of a Boyden chamber. The ECs were washed, fixed, stained, and imaged by fluorescence confocal microscopy in a plane through the center of the pore. (A) EC actin visualized by staining with Alexa Fluor 488phalloidin. Cells are attached to the upper face (U) of the filter. A large cellular protrusion into a filter pore (white arrow) has reached the lower face of the filter (L). (B) Caveolin-1 was visualized by fluorescence confocal microscopy by using rabbit anti-human caveolin-1 as primary antibody followed by biotinylated goat anti-rabbit antibody and Texas Red-avidin. A three-dimensional reconstruction of caveolin-1 immunofluorescence in a transmigrating EC was generated from a series of xz-views. The front is tilted downward by 20° to facilitate visualization of the pore in the lower face of the filter (white arrow). (C–E) Individual xz-views of the cell in B at 2.25-μm intervals. (F and G) Immunofluorescence images (xz-views) of caveolin-1 in other transmigrating ECs from the same experiment. (H) Immunofluorescence image of a transmigrating cell from the same experiment, by using nonimmune, rabbit IgG instead of anti-caveolin-1 as an immunostaining control.
    Figure Legend Snippet: Caveolin-1 accumulates in the forward extension of transmigrating ECs. ECs were allowed to migrate for 2 h across a collagen-coated polycarbonate filter (8-μm pore size) toward 10 ng ml – 1 basic FGF in the lower compartment of a Boyden chamber. The ECs were washed, fixed, stained, and imaged by fluorescence confocal microscopy in a plane through the center of the pore. (A) EC actin visualized by staining with Alexa Fluor 488phalloidin. Cells are attached to the upper face (U) of the filter. A large cellular protrusion into a filter pore (white arrow) has reached the lower face of the filter (L). (B) Caveolin-1 was visualized by fluorescence confocal microscopy by using rabbit anti-human caveolin-1 as primary antibody followed by biotinylated goat anti-rabbit antibody and Texas Red-avidin. A three-dimensional reconstruction of caveolin-1 immunofluorescence in a transmigrating EC was generated from a series of xz-views. The front is tilted downward by 20° to facilitate visualization of the pore in the lower face of the filter (white arrow). (C–E) Individual xz-views of the cell in B at 2.25-μm intervals. (F and G) Immunofluorescence images (xz-views) of caveolin-1 in other transmigrating ECs from the same experiment. (H) Immunofluorescence image of a transmigrating cell from the same experiment, by using nonimmune, rabbit IgG instead of anti-caveolin-1 as an immunostaining control.

    Techniques Used: Staining, Fluorescence, Confocal Microscopy, Avidin-Biotin Assay, Immunofluorescence, Generated, Immunostaining

    10) Product Images from "Heterogeneous induction of microglia M2a phenotype by central administration of interleukin-4"

    Article Title: Heterogeneous induction of microglia M2a phenotype by central administration of interleukin-4

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-014-0211-6

    Selective induction of Arg1 expression in a subset of microglia. Brains after 16 h intracerebroventricular treatment with vehicle (A , C) and IL4 (B , D) were formalin fixed and embedded in paraffin and analyzed by immunohistochemistry to visualize Iba1 (A , B) or Arg1 (C , D) in the frontal cortex. Arg1 expression is observed selectively in microglia-like cells after IL4 treatment and is restricted to a subset of cells (D) . Scale bar 100 μm. The results obtained by counting the number of Arg1-positive microglia cells and the total number of Iba1-positive microglia cells demonstrated that an ≈ 35% subset of microglia cells is Arg1 responder microglia (E) . Veh., vehicle.
    Figure Legend Snippet: Selective induction of Arg1 expression in a subset of microglia. Brains after 16 h intracerebroventricular treatment with vehicle (A , C) and IL4 (B , D) were formalin fixed and embedded in paraffin and analyzed by immunohistochemistry to visualize Iba1 (A , B) or Arg1 (C , D) in the frontal cortex. Arg1 expression is observed selectively in microglia-like cells after IL4 treatment and is restricted to a subset of cells (D) . Scale bar 100 μm. The results obtained by counting the number of Arg1-positive microglia cells and the total number of Iba1-positive microglia cells demonstrated that an ≈ 35% subset of microglia cells is Arg1 responder microglia (E) . Veh., vehicle.

    Techniques Used: Expressing, Immunohistochemistry

    Time course of IL4-induced M2 gene expression in frontal cortex and striatum . Following the indicated time intervals after intracerebroventricular injection of saline (Veh) or IL4, the RNA extracted from the frontal cortex (Fr cortex) and striatum was analyzed by real time PCR to evaluate (A) Fizz1 , (B) Arg1 , and (C) Ym1 gene expression. Data sets for each gene were calculated using the 2 -ddCt method with respect to the mean value of the 8 h vehicle group. Bars represent mean values ± SEM. * P
    Figure Legend Snippet: Time course of IL4-induced M2 gene expression in frontal cortex and striatum . Following the indicated time intervals after intracerebroventricular injection of saline (Veh) or IL4, the RNA extracted from the frontal cortex (Fr cortex) and striatum was analyzed by real time PCR to evaluate (A) Fizz1 , (B) Arg1 , and (C) Ym1 gene expression. Data sets for each gene were calculated using the 2 -ddCt method with respect to the mean value of the 8 h vehicle group. Bars represent mean values ± SEM. * P

    Techniques Used: Expressing, Injection, Real-time Polymerase Chain Reaction

    11) Product Images from "Synergistic Neurotoxicity of Opioids and Human Immunodeficiency Virus-1 Tat Protein in Striatal Neurons In Vitro"

    Article Title: Synergistic Neurotoxicity of Opioids and Human Immunodeficiency Virus-1 Tat Protein in Striatal Neurons In Vitro

    Journal: Neuroscience

    doi:

    Characterization of neural cells in mouse striatal cultures (A-G). (A) Phase contrast photomicrograph of striatal cells at 5-7 days in vitro . A majority of the cells are neurons; scale bar = 20 μm. (B) Astrocytes were characterized by large nuclei with multiple nucleoli and dispersed heterochromatin (Hoechst 33342 blue immunofluorescence) and glial fibrillary acidic (GFAP) immunofluorescence (Cy3 red fluorochrome); scale bar = 25 μm. (C-E) Subpopulations of striatal neurons possessed μ (C), κ (E), and to a lesser extent δ (not shown), opioid receptor immunoreactivity. D E show phase contrast and κ opioid receptor immunofluorescent images of the same cells; scale bar = 20 μm. (F-G) Triple-label-identification of neurons, astrocytes and microglia in striatal cultures. Neurons were identified using anti-PGP 9.5 indirect immunofluorescence (Alexa 488, green product), while astrocytes were labeled using anti-GFAP indirect immunofluorescence (Alexa 546, red product) and microglia were detected using IB 4 biotinylated isolectin-conjugated (via avidin) to Alexa 350 (blue fluorescent product) (arrow) (F). A majority of the cells were neurons; microglia comprised 0.7% of the total cells. (G) A phase-contrast photomicrograph of the same cells as in Fig. 1F, some of which were non-viable/degenerating (hatched arrows); PGP 9.5 immunoreactivity is retained by many degenerating neurons; scale bar = 20 μm.
    Figure Legend Snippet: Characterization of neural cells in mouse striatal cultures (A-G). (A) Phase contrast photomicrograph of striatal cells at 5-7 days in vitro . A majority of the cells are neurons; scale bar = 20 μm. (B) Astrocytes were characterized by large nuclei with multiple nucleoli and dispersed heterochromatin (Hoechst 33342 blue immunofluorescence) and glial fibrillary acidic (GFAP) immunofluorescence (Cy3 red fluorochrome); scale bar = 25 μm. (C-E) Subpopulations of striatal neurons possessed μ (C), κ (E), and to a lesser extent δ (not shown), opioid receptor immunoreactivity. D E show phase contrast and κ opioid receptor immunofluorescent images of the same cells; scale bar = 20 μm. (F-G) Triple-label-identification of neurons, astrocytes and microglia in striatal cultures. Neurons were identified using anti-PGP 9.5 indirect immunofluorescence (Alexa 488, green product), while astrocytes were labeled using anti-GFAP indirect immunofluorescence (Alexa 546, red product) and microglia were detected using IB 4 biotinylated isolectin-conjugated (via avidin) to Alexa 350 (blue fluorescent product) (arrow) (F). A majority of the cells were neurons; microglia comprised 0.7% of the total cells. (G) A phase-contrast photomicrograph of the same cells as in Fig. 1F, some of which were non-viable/degenerating (hatched arrows); PGP 9.5 immunoreactivity is retained by many degenerating neurons; scale bar = 20 μm.

    Techniques Used: In Vitro, Immunofluorescence, Labeling, Avidin-Biotin Assay

    12) Product Images from "Selective Restoration of Pomc Expression in Glutamatergic POMC Neurons: Evidence for a Dynamic Hypothalamic Neurotransmitter Network"

    Article Title: Selective Restoration of Pomc Expression in Glutamatergic POMC Neurons: Evidence for a Dynamic Hypothalamic Neurotransmitter Network

    Journal: eNeuro

    doi: 10.1523/ENEURO.0400-18.2019

    IHC for POMC cell counts in control and restored mice, and from VGlut2-Cre; tdTomato animals. A , POMC-IR in a male control mouse detected with an Alexa Fluor 568 (red) secondary antibody (1:500). B , POMC-IR in a male restored mouse detected with an Alexa Fluor 568 (red) secondary antibody (1:500). C , POMC-IR in a female control mouse detected with biotinylated secondary antibody (1:500) and visualized with a diaminobenzidine (DAB) reaction (brown). D , POMC-IR in a female restored mouse detected with biotinylated secondary antibody (1:500) and visualized with a DAB reaction (brown). E , POMC-IR in a female Vglut2-Cre; tdTomato mouse detected with an Alexa Fluor 488 (green) secondary antibody (1:500; mirrored section from Fig. 1 G , H ). F , POMC neuron cell counts from sections (three per mouse). There was no difference between control (blue bars) or restored (green bars) mice, but only in the method of secondary labeling used. Male data for each group represented by filled blue circles and female data shown by filled pink circles; **** p
    Figure Legend Snippet: IHC for POMC cell counts in control and restored mice, and from VGlut2-Cre; tdTomato animals. A , POMC-IR in a male control mouse detected with an Alexa Fluor 568 (red) secondary antibody (1:500). B , POMC-IR in a male restored mouse detected with an Alexa Fluor 568 (red) secondary antibody (1:500). C , POMC-IR in a female control mouse detected with biotinylated secondary antibody (1:500) and visualized with a diaminobenzidine (DAB) reaction (brown). D , POMC-IR in a female restored mouse detected with biotinylated secondary antibody (1:500) and visualized with a DAB reaction (brown). E , POMC-IR in a female Vglut2-Cre; tdTomato mouse detected with an Alexa Fluor 488 (green) secondary antibody (1:500; mirrored section from Fig. 1 G , H ). F , POMC neuron cell counts from sections (three per mouse). There was no difference between control (blue bars) or restored (green bars) mice, but only in the method of secondary labeling used. Male data for each group represented by filled blue circles and female data shown by filled pink circles; **** p

    Techniques Used: Immunohistochemistry, Mouse Assay, Labeling

    13) Product Images from "Characterization of G-Protein-Gated K+ Channels Composed of Kir3.2 Subunits in Dopaminergic Neurons of the Substantia Nigra"

    Article Title: Characterization of G-Protein-Gated K+ Channels Composed of Kir3.2 Subunits in Dopaminergic Neurons of the Substantia Nigra

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.19-03-01006.1999

    A , RT-PCR analysis of Kir3.0 mRNAs in rat SN and Cx. Using the Kir3.0-specific primers indicated above each panel, their expression was examined in cDNAs obtained from rat SN ( odd numbers ) and Cx ( even numbers ). PCR products (519, 369, 456, and 574 bp) of Kir3.2a, Kir3.2c, Kir3.3, and Kir3.2b were detected in both SN and Cx, whereas that of Kir3.1 (666 bp) was found in Cx but not in SN. Numbers indicate the standard markers in base pairs. B , Immunoprecipitation analysis of Kir3.2 isoforms in rat SN and Cx. Biotinylated membrane proteins of rat SN ( a ) or Cx ( b ) were incubated with aG2C-3 ( lanes 1 - 3 ) or aG2A-5 antibodies ( lanes 4 , 5 ) as indicated. The immunoprecipitants were detected with streptavidin–HRP ( SA - HRP ; lanes 1 , 4 ), aG2A-5 ( lane 2 ), aG1C-1 ( lane 3 ), or aG2C-3 antibodies ( lane 5 ). The positions of Kir3.2a ( small open arrowheads ) and Kir3.2c ( large arrowheads ) isoforms and Kir3.1 subunits ( arrows ) are indicated. IgG heavy chains or unknown bands are indicated with asterisks and number signs , respectively. Numbers on the left of the panels indicate the molecular weights of the standard markers in kilodaltons. C , Detection of Kir3.3 in the aG2A-5 immunoprecipitant from rat Cx. The lysates of HEK293T cells transfected with the plasmids indicated in each lane were examined for specificity of aG3NC and aG2B-2 with immunoblot analysis ( a ). Both antibodies could specifically detect proteins at 41 kDa of Kir3.3 and 38 kDa of Kir3.2b, respectively. When the PVDF membranes blotted with SA–HRP were overexposed to the films until the bands of Kir3.2a and Kir3.2c were indistinguishable ( b ), a faint signal at 40 kDa ( small arrows ) was detected in the immunocomplex of aG2A-5 obtained from Cx ( lane 2 ), not from SN ( lane 1 ). The 40 kDa protein in the immunoprecipitant has an immunoreactivity to aG3NC. However, signal with aG2B-2 could not be found either in SN or in Cx.
    Figure Legend Snippet: A , RT-PCR analysis of Kir3.0 mRNAs in rat SN and Cx. Using the Kir3.0-specific primers indicated above each panel, their expression was examined in cDNAs obtained from rat SN ( odd numbers ) and Cx ( even numbers ). PCR products (519, 369, 456, and 574 bp) of Kir3.2a, Kir3.2c, Kir3.3, and Kir3.2b were detected in both SN and Cx, whereas that of Kir3.1 (666 bp) was found in Cx but not in SN. Numbers indicate the standard markers in base pairs. B , Immunoprecipitation analysis of Kir3.2 isoforms in rat SN and Cx. Biotinylated membrane proteins of rat SN ( a ) or Cx ( b ) were incubated with aG2C-3 ( lanes 1 - 3 ) or aG2A-5 antibodies ( lanes 4 , 5 ) as indicated. The immunoprecipitants were detected with streptavidin–HRP ( SA - HRP ; lanes 1 , 4 ), aG2A-5 ( lane 2 ), aG1C-1 ( lane 3 ), or aG2C-3 antibodies ( lane 5 ). The positions of Kir3.2a ( small open arrowheads ) and Kir3.2c ( large arrowheads ) isoforms and Kir3.1 subunits ( arrows ) are indicated. IgG heavy chains or unknown bands are indicated with asterisks and number signs , respectively. Numbers on the left of the panels indicate the molecular weights of the standard markers in kilodaltons. C , Detection of Kir3.3 in the aG2A-5 immunoprecipitant from rat Cx. The lysates of HEK293T cells transfected with the plasmids indicated in each lane were examined for specificity of aG3NC and aG2B-2 with immunoblot analysis ( a ). Both antibodies could specifically detect proteins at 41 kDa of Kir3.3 and 38 kDa of Kir3.2b, respectively. When the PVDF membranes blotted with SA–HRP were overexposed to the films until the bands of Kir3.2a and Kir3.2c were indistinguishable ( b ), a faint signal at 40 kDa ( small arrows ) was detected in the immunocomplex of aG2A-5 obtained from Cx ( lane 2 ), not from SN ( lane 1 ). The 40 kDa protein in the immunoprecipitant has an immunoreactivity to aG3NC. However, signal with aG2B-2 could not be found either in SN or in Cx.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Polymerase Chain Reaction, Immunoprecipitation, Incubation, Transfection

    Characterization of polyclonal antibodies against Kir3.2 isoforms ( A , B ) and immunological analyses of Kir3.2 isoforms expressed in HEK293T cells ( C , D ). A , Alignments of amino acid sequence of Kir3.2 isoforms. The antigens for Kir3.2 isoform-specific antibodies are indicated by boxes at the C termini of the proteins with their amino acid sequences represented in single letter code. The shaded regions M1 , M2 , and H5 indicate the putative transmembrane domains and the pore region of the Kir channel. A putative binding motif for PDZ domain-containing proteins is underlined at the C terminus of Kir3.2c. B , Immunofluorescent images of Kir3.2 isoforms obtained with Kir3.2-specific antibodies. HEK293T cells were transfected with Kir3.2a ( a , d , g ) or Kir3.2c ( b , c , e , f , h , i ). After 2 d, the products in cells were immunostained with aG2A-5 ( a – c ), aG2C-3 ( d – f ), and aGIRK2 ( g – i ) antibodies and analyzed with confocal microscopy. Immunoreactivity to each antibody was indicated with green images superimposed with nuclei staining with propidium iodide ( red ). Immunolabeling was blocked by preincubation with their antigenic peptides ( c , f ) or C terminal of mouse GIRK2 (amino acids 374–414) fused with glutathione S -transferase ( i ). Scale bar, 30 μm. C , Immunoblot analysis of Kir3.2 isoforms. Membrane fractions obtained from Kir3.2a- ( lane 1 ), Kir3.2c- ( lane 2 ), or both Kir3.2a- and Kir3.2c- ( lane 3 ) transfected HEK293T cells and the mixture of Kir3.2a and Kir3.2c membranes ( lane 4 ) were separated with SDS-PAGE, transferred to PVDF membranes, and then immunoblotted with aG2A-5 ( top panel ) or aG2C-3 ( bottom panel ) antibodies. D , Immunoprecipitation analysis of Kir3.2 isoforms. Kir3.2 isoforms in the membrane fraction of the cells were solubilized and isolated with aG2A-5 and aG2C-3 antibodies ( top and bottom panels , respectively). Each Kir3.2 isoform was biotinylated before extraction from the membranes. Biotinylated Kir3.2 isoforms were detected with the incubation of streptavidin–HRP conjugate. Migration positions of Kir3.2a and Kir3.2c on the gels are indicated on the right side of each panel .
    Figure Legend Snippet: Characterization of polyclonal antibodies against Kir3.2 isoforms ( A , B ) and immunological analyses of Kir3.2 isoforms expressed in HEK293T cells ( C , D ). A , Alignments of amino acid sequence of Kir3.2 isoforms. The antigens for Kir3.2 isoform-specific antibodies are indicated by boxes at the C termini of the proteins with their amino acid sequences represented in single letter code. The shaded regions M1 , M2 , and H5 indicate the putative transmembrane domains and the pore region of the Kir channel. A putative binding motif for PDZ domain-containing proteins is underlined at the C terminus of Kir3.2c. B , Immunofluorescent images of Kir3.2 isoforms obtained with Kir3.2-specific antibodies. HEK293T cells were transfected with Kir3.2a ( a , d , g ) or Kir3.2c ( b , c , e , f , h , i ). After 2 d, the products in cells were immunostained with aG2A-5 ( a – c ), aG2C-3 ( d – f ), and aGIRK2 ( g – i ) antibodies and analyzed with confocal microscopy. Immunoreactivity to each antibody was indicated with green images superimposed with nuclei staining with propidium iodide ( red ). Immunolabeling was blocked by preincubation with their antigenic peptides ( c , f ) or C terminal of mouse GIRK2 (amino acids 374–414) fused with glutathione S -transferase ( i ). Scale bar, 30 μm. C , Immunoblot analysis of Kir3.2 isoforms. Membrane fractions obtained from Kir3.2a- ( lane 1 ), Kir3.2c- ( lane 2 ), or both Kir3.2a- and Kir3.2c- ( lane 3 ) transfected HEK293T cells and the mixture of Kir3.2a and Kir3.2c membranes ( lane 4 ) were separated with SDS-PAGE, transferred to PVDF membranes, and then immunoblotted with aG2A-5 ( top panel ) or aG2C-3 ( bottom panel ) antibodies. D , Immunoprecipitation analysis of Kir3.2 isoforms. Kir3.2 isoforms in the membrane fraction of the cells were solubilized and isolated with aG2A-5 and aG2C-3 antibodies ( top and bottom panels , respectively). Each Kir3.2 isoform was biotinylated before extraction from the membranes. Biotinylated Kir3.2 isoforms were detected with the incubation of streptavidin–HRP conjugate. Migration positions of Kir3.2a and Kir3.2c on the gels are indicated on the right side of each panel .

    Techniques Used: Sequencing, Binding Assay, Transfection, Confocal Microscopy, Staining, Immunolabeling, SDS Page, Immunoprecipitation, Isolation, Incubation, Migration

    14) Product Images from "Characterization of G-Protein-Gated K+ Channels Composed of Kir3.2 Subunits in Dopaminergic Neurons of the Substantia Nigra"

    Article Title: Characterization of G-Protein-Gated K+ Channels Composed of Kir3.2 Subunits in Dopaminergic Neurons of the Substantia Nigra

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.19-03-01006.1999

    A , RT-PCR analysis of Kir3.0 mRNAs in rat SN and Cx. Using the Kir3.0-specific primers indicated above each panel, their expression was examined in cDNAs obtained from rat SN ( odd numbers ) and Cx ( even numbers ). PCR products (519, 369, 456, and 574 bp) of Kir3.2a, Kir3.2c, Kir3.3, and Kir3.2b were detected in both SN and Cx, whereas that of Kir3.1 (666 bp) was found in Cx but not in SN. Numbers indicate the standard markers in base pairs. B , Immunoprecipitation analysis of Kir3.2 isoforms in rat SN and Cx. Biotinylated membrane proteins of rat SN ( a ) or Cx ( b ) were incubated with aG2C-3 ( lanes 1 - 3 ) or aG2A-5 antibodies ( lanes 4 , 5 ) as indicated. The immunoprecipitants were detected with streptavidin–HRP ( SA - HRP ; lanes 1 , 4 ), aG2A-5 ( lane 2 ), aG1C-1 ( lane 3 ), or aG2C-3 antibodies ( lane 5 ). The positions of Kir3.2a ( small open arrowheads ) and Kir3.2c ( large arrowheads ) isoforms and Kir3.1 subunits ( arrows ) are indicated. IgG heavy chains or unknown bands are indicated with asterisks and number signs , respectively. Numbers on the left of the panels indicate the molecular weights of the standard markers in kilodaltons. C , Detection of Kir3.3 in the aG2A-5 immunoprecipitant from rat Cx. The lysates of HEK293T cells transfected with the plasmids indicated in each lane were examined for specificity of aG3NC and aG2B-2 with immunoblot analysis ( a ). Both antibodies could specifically detect proteins at 41 kDa of Kir3.3 and 38 kDa of Kir3.2b, respectively. When the PVDF membranes blotted with SA–HRP were overexposed to the films until the bands of Kir3.2a and Kir3.2c were indistinguishable ( b ), a faint signal at 40 kDa ( small arrows ) was detected in the immunocomplex of aG2A-5 obtained from Cx ( lane 2 ), not from SN ( lane 1 ). The 40 kDa protein in the immunoprecipitant has an immunoreactivity to aG3NC. However, signal with aG2B-2 could not be found either in SN or in Cx.
    Figure Legend Snippet: A , RT-PCR analysis of Kir3.0 mRNAs in rat SN and Cx. Using the Kir3.0-specific primers indicated above each panel, their expression was examined in cDNAs obtained from rat SN ( odd numbers ) and Cx ( even numbers ). PCR products (519, 369, 456, and 574 bp) of Kir3.2a, Kir3.2c, Kir3.3, and Kir3.2b were detected in both SN and Cx, whereas that of Kir3.1 (666 bp) was found in Cx but not in SN. Numbers indicate the standard markers in base pairs. B , Immunoprecipitation analysis of Kir3.2 isoforms in rat SN and Cx. Biotinylated membrane proteins of rat SN ( a ) or Cx ( b ) were incubated with aG2C-3 ( lanes 1 - 3 ) or aG2A-5 antibodies ( lanes 4 , 5 ) as indicated. The immunoprecipitants were detected with streptavidin–HRP ( SA - HRP ; lanes 1 , 4 ), aG2A-5 ( lane 2 ), aG1C-1 ( lane 3 ), or aG2C-3 antibodies ( lane 5 ). The positions of Kir3.2a ( small open arrowheads ) and Kir3.2c ( large arrowheads ) isoforms and Kir3.1 subunits ( arrows ) are indicated. IgG heavy chains or unknown bands are indicated with asterisks and number signs , respectively. Numbers on the left of the panels indicate the molecular weights of the standard markers in kilodaltons. C , Detection of Kir3.3 in the aG2A-5 immunoprecipitant from rat Cx. The lysates of HEK293T cells transfected with the plasmids indicated in each lane were examined for specificity of aG3NC and aG2B-2 with immunoblot analysis ( a ). Both antibodies could specifically detect proteins at 41 kDa of Kir3.3 and 38 kDa of Kir3.2b, respectively. When the PVDF membranes blotted with SA–HRP were overexposed to the films until the bands of Kir3.2a and Kir3.2c were indistinguishable ( b ), a faint signal at 40 kDa ( small arrows ) was detected in the immunocomplex of aG2A-5 obtained from Cx ( lane 2 ), not from SN ( lane 1 ). The 40 kDa protein in the immunoprecipitant has an immunoreactivity to aG3NC. However, signal with aG2B-2 could not be found either in SN or in Cx.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Polymerase Chain Reaction, Immunoprecipitation, Incubation, Transfection

    15) Product Images from "Prevalent Overexpression of Prolyl Isomerase Pin1 in Human Cancers"

    Article Title: Prevalent Overexpression of Prolyl Isomerase Pin1 in Human Cancers

    Journal: The American Journal of Pathology

    doi:

    Immunocytochemical staining of Pin1 in cultured cells. Cells were incubated with anti-Pin1 antibodies, followed by secondary biotinylated antibody and ABC (avidin and biotinylated horseradish peroxidase complex)-diaminobenzidine reaction. A: WI38 (normal
    Figure Legend Snippet: Immunocytochemical staining of Pin1 in cultured cells. Cells were incubated with anti-Pin1 antibodies, followed by secondary biotinylated antibody and ABC (avidin and biotinylated horseradish peroxidase complex)-diaminobenzidine reaction. A: WI38 (normal

    Techniques Used: Staining, Cell Culture, Incubation, Avidin-Biotin Assay

    16) Product Images from "Phosphoinositide 3-Kinase ? Regulates Airway Smooth Muscle Contraction by Modulating Calcium Oscillations"

    Article Title: Phosphoinositide 3-Kinase ? Regulates Airway Smooth Muscle Contraction by Modulating Calcium Oscillations

    Journal: The Journal of Pharmacology and Experimental Therapeutics

    doi: 10.1124/jpet.110.168518

    Expression of PI3Kγ protein in mouse ASM cells. A, representative immunohistochemical staining of PI3Kγ protein in mouse trachea (a) and lung slices (c) using a PI3Kγ-specific antibody or isotype-specific negative control antibody (rabbit IgG) in a parallel section of trachea (b) and lung slices (d). PI3Kγ staining was localized in ASM and epithelium but not in cartilage. All the sections were counterstained with blue hematoxylin. B, Western blot analysis of PI3Kγ protein in lysates of mouse lung tissue, heart tissue, and isolated mouse ASM cells using a PI3Kγ-specific antibody. Images shown are representative of four independent experiments. β-Actin was used as a loading control. C, immunofluorescent staining of PI3Kγ protein (green) in cultured mouse ASM cells, and staining of α-actin (red) as a marker of smooth muscle cells.
    Figure Legend Snippet: Expression of PI3Kγ protein in mouse ASM cells. A, representative immunohistochemical staining of PI3Kγ protein in mouse trachea (a) and lung slices (c) using a PI3Kγ-specific antibody or isotype-specific negative control antibody (rabbit IgG) in a parallel section of trachea (b) and lung slices (d). PI3Kγ staining was localized in ASM and epithelium but not in cartilage. All the sections were counterstained with blue hematoxylin. B, Western blot analysis of PI3Kγ protein in lysates of mouse lung tissue, heart tissue, and isolated mouse ASM cells using a PI3Kγ-specific antibody. Images shown are representative of four independent experiments. β-Actin was used as a loading control. C, immunofluorescent staining of PI3Kγ protein (green) in cultured mouse ASM cells, and staining of α-actin (red) as a marker of smooth muscle cells.

    Techniques Used: Expressing, Immunohistochemistry, Staining, Negative Control, Western Blot, Isolation, Cell Culture, Marker

    17) Product Images from "Anti-inflammatory and anti-amyloidogenic effects of a small molecule, 2,4-bis(p-hydroxyphenyl)-2-butenal in Tg2576 Alzheimer's disease mice model"

    Article Title: Anti-inflammatory and anti-amyloidogenic effects of a small molecule, 2,4-bis(p-hydroxyphenyl)-2-butenal in Tg2576 Alzheimer's disease mice model

    Journal: Journal of Neuroinflammation

    doi: 10.1186/1742-2094-10-2

    Inhibitory effects of HPB242 on accumulation of Aβ 1-42 in the brain of Tg2576 mice. Aβ accumulation in the brains of Tg2576 mice was determined by immunohistochemical analysis using Aβ 1-42 -specific antibody ( A ). The sections of Tg2576 mouse brains were incubated with anti-Aβ 1-42 primary antibody, and biotinylated secondary antibody. Immunoperoxidase staining of brains of Tg2576 and treated-Tg2576 mice shown (brown color). Aβ 1-42 level was measured in mouse brains by ELISA as described in Materials and Methods ( B ). The value is mean ± standard error of the mean (SEM) (n = 8 mice). The activity of β-secretase was investigated using assay kit as described ( C ). Values measured from each group of mice were calibrated by the amount of protein and expressed as mean ± SEM (n = 8 mice). The expression of APP and BACE1 were detected by western blotting using specific antibodies in the mouse brain. Each blot is representative of three experiments ( D ). *Significantly different from non-treated Tg2576 mice ( P
    Figure Legend Snippet: Inhibitory effects of HPB242 on accumulation of Aβ 1-42 in the brain of Tg2576 mice. Aβ accumulation in the brains of Tg2576 mice was determined by immunohistochemical analysis using Aβ 1-42 -specific antibody ( A ). The sections of Tg2576 mouse brains were incubated with anti-Aβ 1-42 primary antibody, and biotinylated secondary antibody. Immunoperoxidase staining of brains of Tg2576 and treated-Tg2576 mice shown (brown color). Aβ 1-42 level was measured in mouse brains by ELISA as described in Materials and Methods ( B ). The value is mean ± standard error of the mean (SEM) (n = 8 mice). The activity of β-secretase was investigated using assay kit as described ( C ). Values measured from each group of mice were calibrated by the amount of protein and expressed as mean ± SEM (n = 8 mice). The expression of APP and BACE1 were detected by western blotting using specific antibodies in the mouse brain. Each blot is representative of three experiments ( D ). *Significantly different from non-treated Tg2576 mice ( P

    Techniques Used: Mouse Assay, Immunohistochemistry, Incubation, Immunoperoxidase Staining, Enzyme-linked Immunosorbent Assay, Activity Assay, Expressing, Western Blot

    HPB242 inhibited activation of astrocytes and microglia, and reduced expression of COX-2 and iNOS in the brains of Tg2576 mice. The effect of HPB242 on reactive astrocytes and activated microglia cells was measured by immunohistochemical analysis and western blotting analysis. The sections of mice brain incubated with anti-glial fibrillary acidic protein (GFAP) ( A ) or ionized calcium binding adaptor molecule 1 (Iba1) ( B ) primary antibody and the biotinylated secondary antibody (n = 8). The stained tissues were viewed with a microscope (×100 or 400). Expression of GFAP and Iba1 were also examined by specific antibodies in the cortex and hippocampus ( C ). Each blot is representative for three experiments (n = 8). Inhibitory effects of HPB242 on the Tg2576 mice brain expression of inflammatory proteins. The sections of mouse brain incubated with anti-iNOS ( D ) or COX-2 ( E ) and the biotinylated secondary antibody (n=8). The resulting tissue was viewed with a microscope (×100 or 400). The expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were detected by western blotting using specific antibodies ( F ). Each blot is representative for three experiments (n = 8). *Significantly different to non-treated Tg2576 mice ( P
    Figure Legend Snippet: HPB242 inhibited activation of astrocytes and microglia, and reduced expression of COX-2 and iNOS in the brains of Tg2576 mice. The effect of HPB242 on reactive astrocytes and activated microglia cells was measured by immunohistochemical analysis and western blotting analysis. The sections of mice brain incubated with anti-glial fibrillary acidic protein (GFAP) ( A ) or ionized calcium binding adaptor molecule 1 (Iba1) ( B ) primary antibody and the biotinylated secondary antibody (n = 8). The stained tissues were viewed with a microscope (×100 or 400). Expression of GFAP and Iba1 were also examined by specific antibodies in the cortex and hippocampus ( C ). Each blot is representative for three experiments (n = 8). Inhibitory effects of HPB242 on the Tg2576 mice brain expression of inflammatory proteins. The sections of mouse brain incubated with anti-iNOS ( D ) or COX-2 ( E ) and the biotinylated secondary antibody (n=8). The resulting tissue was viewed with a microscope (×100 or 400). The expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were detected by western blotting using specific antibodies ( F ). Each blot is representative for three experiments (n = 8). *Significantly different to non-treated Tg2576 mice ( P

    Techniques Used: Activation Assay, Expressing, Mouse Assay, Immunohistochemistry, Western Blot, Incubation, Binding Assay, Staining, Microscopy

    18) Product Images from "Frequency-Dependent Release of Substance P Mediates Heterosynaptic Potentiation of Glutamatergic Synaptic Responses in the Rat Visual Thalamus"

    Article Title: Frequency-Dependent Release of Substance P Mediates Heterosynaptic Potentiation of Glutamatergic Synaptic Responses in the Rat Visual Thalamus

    Journal: Journal of Neurophysiology

    doi: 10.1152/jn.00010.2010

    Tecto-LPN terminals contain SP. A : a large injection of biotinylated dextran amine (BDA) was made in the superficial layers of the superior colliculus (SC) to label axons in the LPN by anterograde transport. LPN tissue was subsequently treated to reveal
    Figure Legend Snippet: Tecto-LPN terminals contain SP. A : a large injection of biotinylated dextran amine (BDA) was made in the superficial layers of the superior colliculus (SC) to label axons in the LPN by anterograde transport. LPN tissue was subsequently treated to reveal

    Techniques Used: Injection

    19) Product Images from "In vivo antioxidant treatment protects against bleomycin-induced lung damage in rats"

    Article Title: In vivo antioxidant treatment protects against bleomycin-induced lung damage in rats

    Journal: British Journal of Pharmacology

    doi: 10.1038/sj.bjp.0705138

    Immunohistochemical analysis of NF- κ B in lung sections obtained at 3 days postbleomycin or saline exposure in vehicle- or N -acetylcysteine (NAC, 300 mg kg −1 per day, i.p.)-treated rats. Lung sections were obtained from control rats (vehicle+vehicle; (a, b)), and from rats exposed to bleomycin and receiving drug vehicle (c, d) or NAC (e, f). Panels show sections stained with anti-p65 antibody followed by the avidin–biotin complex. The immunohistochemical localization of NF- κ B appears as dark-brown positive nuclear staining in airway epithelial cells as well as macrophages and other inflammatory cells like polymorphonuclear leukocytes in interalveolar septa. There is a low staining level in control lungs. Note the high level of staining in the epithelium of bleomycin-exposed rats (panel (d) compared to the virtual absence in epithelial cells from control animals (panel (b). The increased expression of p65 in vehicle+bleomycin rats was markedly diminished in NAC+bleomycin samples (cf. (c,d) and (e,f)). Original magnification × 25 (a, c, e) and × 40 (b, d, f).
    Figure Legend Snippet: Immunohistochemical analysis of NF- κ B in lung sections obtained at 3 days postbleomycin or saline exposure in vehicle- or N -acetylcysteine (NAC, 300 mg kg −1 per day, i.p.)-treated rats. Lung sections were obtained from control rats (vehicle+vehicle; (a, b)), and from rats exposed to bleomycin and receiving drug vehicle (c, d) or NAC (e, f). Panels show sections stained with anti-p65 antibody followed by the avidin–biotin complex. The immunohistochemical localization of NF- κ B appears as dark-brown positive nuclear staining in airway epithelial cells as well as macrophages and other inflammatory cells like polymorphonuclear leukocytes in interalveolar septa. There is a low staining level in control lungs. Note the high level of staining in the epithelium of bleomycin-exposed rats (panel (d) compared to the virtual absence in epithelial cells from control animals (panel (b). The increased expression of p65 in vehicle+bleomycin rats was markedly diminished in NAC+bleomycin samples (cf. (c,d) and (e,f)). Original magnification × 25 (a, c, e) and × 40 (b, d, f).

    Techniques Used: Immunohistochemistry, Staining, Avidin-Biotin Assay, Expressing

    20) Product Images from "Reawakening Retrocyclins: Ancestral Human Defensins Active Against HIV-1Rousing a Latent Defense Mechanism to Fight HIV"

    Article Title: Reawakening Retrocyclins: Ancestral Human Defensins Active Against HIV-1Rousing a Latent Defense Mechanism to Fight HIV

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.1000095

    Expression of Retrocyclins in Cervicovaginal Tissue Model Using Aminoglycosides (A) Cervicovaginal tissues were treated with PBS (Con) or 10 μl tobramycin (Tob) and incubated with rabbit preimmune serum or antiretrocyclin antibody. The slides were then incubated with biotinylated goat anti-rabbit IgG secondary antibody and then stained using FITC-avidin. (B) Cytotoxicity was determined by measuring LDH activity in media underlying the tissues treated with PBS or tobramycin as indicated. Bars represent absorbance measured as 490 nm and error bars represent SEM; n = 6. (C) HPLC trace of extracts of tissues treated with 10 μg/ml tobramycin (tissue + Tob) and 20 μg of synthetic RC-100. (D) RC-100 synthetic peptide (indicated amounts), HPLC fractions 27–29 of control, tobramycin-treated, and RC-100 were dotted on a PVDF membrane and analyzed by immuno-dotblot.
    Figure Legend Snippet: Expression of Retrocyclins in Cervicovaginal Tissue Model Using Aminoglycosides (A) Cervicovaginal tissues were treated with PBS (Con) or 10 μl tobramycin (Tob) and incubated with rabbit preimmune serum or antiretrocyclin antibody. The slides were then incubated with biotinylated goat anti-rabbit IgG secondary antibody and then stained using FITC-avidin. (B) Cytotoxicity was determined by measuring LDH activity in media underlying the tissues treated with PBS or tobramycin as indicated. Bars represent absorbance measured as 490 nm and error bars represent SEM; n = 6. (C) HPLC trace of extracts of tissues treated with 10 μg/ml tobramycin (tissue + Tob) and 20 μg of synthetic RC-100. (D) RC-100 synthetic peptide (indicated amounts), HPLC fractions 27–29 of control, tobramycin-treated, and RC-100 were dotted on a PVDF membrane and analyzed by immuno-dotblot.

    Techniques Used: Expressing, Incubation, Staining, Avidin-Biotin Assay, Activity Assay, High Performance Liquid Chromatography

    Immunofluorescence Staining of Stably Transfected HL60 Cells Reveals Retrocyclin Peptides (A) Retrocyclin peptides RC-100, RC-101, and RC-101_2K peptides (in duplicates) and (B) RC-100, RC-100b, RC-101, protegrin-1 (PG-1), rhesus theta defensin-1 (RTD-1), and human neutrophil peptides 1–3 (HNP 1–3) were dotted (0–8 ng/4 μl dot) on a PVDF membrane and subjected to immuno-dotblot analysis. (C) VC, R1R3, and A1A3 (100,000 cells each) were fixed onto glass slides and incubated with rabbit anti-RC-101 antibody followed by biotinylated goat anti-rabbit IgG secondary antibody and then stained using fluorescein isothiocyanate (FITC)-avidin. Slides were visualized using Zeiss Axiovert 200M microscope system at 40× magnification. The three rows show FITC staining, DIC, and the merged image, respectively. Scale bar represents 20 μm.
    Figure Legend Snippet: Immunofluorescence Staining of Stably Transfected HL60 Cells Reveals Retrocyclin Peptides (A) Retrocyclin peptides RC-100, RC-101, and RC-101_2K peptides (in duplicates) and (B) RC-100, RC-100b, RC-101, protegrin-1 (PG-1), rhesus theta defensin-1 (RTD-1), and human neutrophil peptides 1–3 (HNP 1–3) were dotted (0–8 ng/4 μl dot) on a PVDF membrane and subjected to immuno-dotblot analysis. (C) VC, R1R3, and A1A3 (100,000 cells each) were fixed onto glass slides and incubated with rabbit anti-RC-101 antibody followed by biotinylated goat anti-rabbit IgG secondary antibody and then stained using fluorescein isothiocyanate (FITC)-avidin. Slides were visualized using Zeiss Axiovert 200M microscope system at 40× magnification. The three rows show FITC staining, DIC, and the merged image, respectively. Scale bar represents 20 μm.

    Techniques Used: Immunofluorescence, Staining, Stable Transfection, Transfection, Incubation, Avidin-Biotin Assay, Microscopy

    21) Product Images from "Co-Regulation of Cell Polarization and Migration by Caveolar Proteins PTRF/Cavin-1 and Caveolin-1"

    Article Title: Co-Regulation of Cell Polarization and Migration by Caveolar Proteins PTRF/Cavin-1 and Caveolin-1

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0043041

    Differential polarization of caveolin-1 and PTRF/cavin-1 during 3D migration. (A) Transmigrating NIH 3T3 cells were fixed and immuno-labeled using anti-caveolin-1 or anti-PTRF/cavin-1 antibodies and imaged by confocal microscopy. Shown are xy planes of cell rear, protrusion through the pore or xz planes through the center of the pore. Bar represents 10 µM. (B) Transmigrating PTRF/cavin-1-GFP expressing PC3 cells were fixed and immuno-labeled using anti-caveolin-1 antibody followed by biotinylated anti-rabbit antibody and texas red avidin. Cells were then imaged by confocal microscopy. Bar represents 10 µM. (C) Schematic representation of the differential caveolin-1 and PTRF/cavin-1 polarization during transmigration.
    Figure Legend Snippet: Differential polarization of caveolin-1 and PTRF/cavin-1 during 3D migration. (A) Transmigrating NIH 3T3 cells were fixed and immuno-labeled using anti-caveolin-1 or anti-PTRF/cavin-1 antibodies and imaged by confocal microscopy. Shown are xy planes of cell rear, protrusion through the pore or xz planes through the center of the pore. Bar represents 10 µM. (B) Transmigrating PTRF/cavin-1-GFP expressing PC3 cells were fixed and immuno-labeled using anti-caveolin-1 antibody followed by biotinylated anti-rabbit antibody and texas red avidin. Cells were then imaged by confocal microscopy. Bar represents 10 µM. (C) Schematic representation of the differential caveolin-1 and PTRF/cavin-1 polarization during transmigration.

    Techniques Used: Migration, Labeling, Confocal Microscopy, Expressing, Avidin-Biotin Assay, Transmigration Assay

    22) Product Images from "Orexin neurons couple neural systems mediating fluid balance with motivation-related circuits"

    Article Title: Orexin neurons couple neural systems mediating fluid balance with motivation-related circuits

    Journal: Behavioral neuroscience

    doi: 10.1037/bne0000250

    Results from Fluorogold and co-injection of BDA and FG infused into the PeF. Injection sites of 2% FG (blue) into the PeF Ox neurons (red) and a co-injection of 1.33% FG (blue) and 2.5% BDA (green) in PeF Ox neurons (red) are presented in panels A and B, respectively. Retrograde labeling was observed along the entirety of the LT (panels C-F). Axon terminals and varicosities (green) were observed in apposition with tyrosine hydroxylase positive neurons in the VTA (red; panels G-J). Putative synaptic contact is indicated by arrows. BDA, biotinylated dextranamine; FG, Fluorogold; Ox, orexin, PeF, Perifornical Area; SFO, subfornical organ.
    Figure Legend Snippet: Results from Fluorogold and co-injection of BDA and FG infused into the PeF. Injection sites of 2% FG (blue) into the PeF Ox neurons (red) and a co-injection of 1.33% FG (blue) and 2.5% BDA (green) in PeF Ox neurons (red) are presented in panels A and B, respectively. Retrograde labeling was observed along the entirety of the LT (panels C-F). Axon terminals and varicosities (green) were observed in apposition with tyrosine hydroxylase positive neurons in the VTA (red; panels G-J). Putative synaptic contact is indicated by arrows. BDA, biotinylated dextranamine; FG, Fluorogold; Ox, orexin, PeF, Perifornical Area; SFO, subfornical organ.

    Techniques Used: Injection, Labeling

    23) Product Images from "The Transcription Factor ETS-1 Mediates Proinflammatory Responses and Neointima Formation in Carotid Artery Endoluminal Vascular Injury"

    Article Title: The Transcription Factor ETS-1 Mediates Proinflammatory Responses and Neointima Formation in Carotid Artery Endoluminal Vascular Injury

    Journal: Hypertension

    doi: 10.1161/HYPERTENSIONAHA.110.150995

    Biotin staining of biotinylated peptide in the vessel. Carotid arteries from Veh-treated rats had background biotin stain that was significantly increased in arteries from ETS-1 DN–treated rats.
    Figure Legend Snippet: Biotin staining of biotinylated peptide in the vessel. Carotid arteries from Veh-treated rats had background biotin stain that was significantly increased in arteries from ETS-1 DN–treated rats.

    Techniques Used: Staining

    24) Product Images from "A novel multiplex assay for simultaneous quantification of total and S129 phosphorylated human alpha-synuclein"

    Article Title: A novel multiplex assay for simultaneous quantification of total and S129 phosphorylated human alpha-synuclein

    Journal: Molecular Neurodegeneration

    doi: 10.1186/s13024-016-0125-0

    Characterization of total and phosphorylated S129 human alpha-synuclein duplex assay . The hook-point for biotinylated 4B12 antibody was determined simultaneously in a mix of Europium Acceptor-bead coupled 11A5 antibody and Terbium Acceptor-bead coupled LB509 antibody ( a ). Arrows indicate optimal 4B12 concentrations for both Acceptor-bead variants. Equivalency of the signal obtained in the total h-asyn assay was demonstrated for pS129 h-asyn and h-asyn proteins in the duplex assay in two consecutive experiments and data plotted in panel ( b ). All protein standards were spiked in naïve rat brain lysate. Lower detection limit (LDL) was 450 pg/ml and is indicated with a dashed line. Intra assay variation was calculated based on three standard curves which were measured on the same day but on separate plates for each emission signal separately ( c and d ). Inter assay variation data was calculated based on six pairs of standard curves performed on separate days ( e and f ). AU arbitrary units
    Figure Legend Snippet: Characterization of total and phosphorylated S129 human alpha-synuclein duplex assay . The hook-point for biotinylated 4B12 antibody was determined simultaneously in a mix of Europium Acceptor-bead coupled 11A5 antibody and Terbium Acceptor-bead coupled LB509 antibody ( a ). Arrows indicate optimal 4B12 concentrations for both Acceptor-bead variants. Equivalency of the signal obtained in the total h-asyn assay was demonstrated for pS129 h-asyn and h-asyn proteins in the duplex assay in two consecutive experiments and data plotted in panel ( b ). All protein standards were spiked in naïve rat brain lysate. Lower detection limit (LDL) was 450 pg/ml and is indicated with a dashed line. Intra assay variation was calculated based on three standard curves which were measured on the same day but on separate plates for each emission signal separately ( c and d ). Inter assay variation data was calculated based on six pairs of standard curves performed on separate days ( e and f ). AU arbitrary units

    Techniques Used: Intra Assay, Inter Assay

    Characterization of total human alpha-synuclein AlphaLISA. The hook-point analysis was evaluated for serial dilutions of biotinylated 4B12 in combination with Europium Acceptor-beads coupled to either LB509 (LB509-Eu) or syn211 (syn211-Eu) using a 19 ng/ml of recombinant GST-tagged h-asyn protein diluted in 10 μg/ml of Tris wild-type rat brain lysate ( a ). Arrow indicates the highest signal obtained and therefore optimal 4B12-biotin concentration to use. Standard curve was established for biotinylated 4B12 and Europium Acceptor-bead coupled LB509 by serial dilution of either GST-tagged h-asyn or mouse asyn (m-asyn) protein spiked in Tris brain lysate obtained from a naïve rat (10 μg/ml) ( b ). The lower detection limit (LDL) was 3.7 pg/ml and is indicated by a dashed line . Intra assay variation was calculated based on two standard curves which were measured on the same day but on separate plates ( c ). Inter assay variation data was calculated based on two pairs of standard curves performed on separate days ( d ). AU arbitrary units
    Figure Legend Snippet: Characterization of total human alpha-synuclein AlphaLISA. The hook-point analysis was evaluated for serial dilutions of biotinylated 4B12 in combination with Europium Acceptor-beads coupled to either LB509 (LB509-Eu) or syn211 (syn211-Eu) using a 19 ng/ml of recombinant GST-tagged h-asyn protein diluted in 10 μg/ml of Tris wild-type rat brain lysate ( a ). Arrow indicates the highest signal obtained and therefore optimal 4B12-biotin concentration to use. Standard curve was established for biotinylated 4B12 and Europium Acceptor-bead coupled LB509 by serial dilution of either GST-tagged h-asyn or mouse asyn (m-asyn) protein spiked in Tris brain lysate obtained from a naïve rat (10 μg/ml) ( b ). The lower detection limit (LDL) was 3.7 pg/ml and is indicated by a dashed line . Intra assay variation was calculated based on two standard curves which were measured on the same day but on separate plates ( c ). Inter assay variation data was calculated based on two pairs of standard curves performed on separate days ( d ). AU arbitrary units

    Techniques Used: Recombinant, Concentration Assay, Serial Dilution, Intra Assay, Inter Assay

    Evaluation of the duplex AlphaLISA performance. Assessment of the Acceptor-bead performance and comparison between Europium and Terbium based beads was carried out once for both the LB509 ( a ) and the 11A5 ( b ) antibodies. In both instances the biotinylated 4B12 antibody was used and signal to background ratio calculated against serial dilutions of h-asyn proteins spiked in naïve rat brain lysate. Presence of any hindrance of Acceptor-bead coupled antibodies against each other was assessed for LB509-Terbium ( c ) and 11A5-Europium beads ( d ). Cross-talk between the channels was assessed using the Resorufine/Amplex Red FP535 Terbium filter ( e ) and the Europium 615 filter ( f ). For these experiments the relevant analyte, Acceptor- and Donor-bead information is given under the x-axis. Both the hindrance of Acceptor-bead coupled antibodies and the channel cross talk were run twice. See Additional file 2 : Figure S2 for a theoretical explanation of the phenomena. AU arbitrary units
    Figure Legend Snippet: Evaluation of the duplex AlphaLISA performance. Assessment of the Acceptor-bead performance and comparison between Europium and Terbium based beads was carried out once for both the LB509 ( a ) and the 11A5 ( b ) antibodies. In both instances the biotinylated 4B12 antibody was used and signal to background ratio calculated against serial dilutions of h-asyn proteins spiked in naïve rat brain lysate. Presence of any hindrance of Acceptor-bead coupled antibodies against each other was assessed for LB509-Terbium ( c ) and 11A5-Europium beads ( d ). Cross-talk between the channels was assessed using the Resorufine/Amplex Red FP535 Terbium filter ( e ) and the Europium 615 filter ( f ). For these experiments the relevant analyte, Acceptor- and Donor-bead information is given under the x-axis. Both the hindrance of Acceptor-bead coupled antibodies and the channel cross talk were run twice. See Additional file 2 : Figure S2 for a theoretical explanation of the phenomena. AU arbitrary units

    Techniques Used:

    Characterization of S129 phosphorylated human alpha-synuclein specific AlphaLISA. The hook-point analysis was evaluated for biotinylated 4B12 and Europium Acceptor-bead conjugated 11A5 ( a ). Pair was tested using 14 ng/ml of pS129 h-asyn protein diluted in 10 μg/ml of Tris brain lysate obtained from an asyn knockout mouse. Arrow indicates the optimal biotinylated antibody concentration to use. Standard curve was established for biotinylated 4B12 and Europium Acceptor-bead coupled 11A5 by serial dilution of either pS129 h-asyn or S129A h-asyn protein spiked in wild-type rodent brain lysate (10 μg/ml) ( b ). The lower detection limit (LDL) was 1.1 pg/ml and is indicated by a dashed line. Intra assay variation was calculated based on two standard curves which were measured on the same day but on separate plates ( c ) while the inter assay variation was calculated based on two pairs of standard curves performed on separate days ( d ). AU arbitrary units
    Figure Legend Snippet: Characterization of S129 phosphorylated human alpha-synuclein specific AlphaLISA. The hook-point analysis was evaluated for biotinylated 4B12 and Europium Acceptor-bead conjugated 11A5 ( a ). Pair was tested using 14 ng/ml of pS129 h-asyn protein diluted in 10 μg/ml of Tris brain lysate obtained from an asyn knockout mouse. Arrow indicates the optimal biotinylated antibody concentration to use. Standard curve was established for biotinylated 4B12 and Europium Acceptor-bead coupled 11A5 by serial dilution of either pS129 h-asyn or S129A h-asyn protein spiked in wild-type rodent brain lysate (10 μg/ml) ( b ). The lower detection limit (LDL) was 1.1 pg/ml and is indicated by a dashed line. Intra assay variation was calculated based on two standard curves which were measured on the same day but on separate plates ( c ) while the inter assay variation was calculated based on two pairs of standard curves performed on separate days ( d ). AU arbitrary units

    Techniques Used: Knock-Out, Concentration Assay, Serial Dilution, Intra Assay, Inter Assay

    25) Product Images from "Pigment Epithelium-Derived Factor (PEDF) mediates cartilage matrix loss in an age-dependent manner under inflammatory conditions"

    Article Title: Pigment Epithelium-Derived Factor (PEDF) mediates cartilage matrix loss in an age-dependent manner under inflammatory conditions

    Journal: BMC Musculoskeletal Disorders

    doi: 10.1186/s12891-017-1410-y

    PEDF-deficiency protects against IL-1β-induced MMP-1, MMP-3 and MMP-13 protein production in metatarsal bone cultures. Metatarsal bones from 10 and 29 week old wild type or PEDF-deficient mice were harvested and cultured in the presence or absence of 10 ng/mL IL-1β for 7 days. Cryosectioned samples were analyzed for MMP protein levels by immunohistochemistry (IHC). AC: articular cartilage. SB: subchondral bone. Scale bar = 100 μm. a Samples were stained with rabbit anti-MMP-1 and counterstained with 0.5% Methyl Green in 0.1 M Sodium Acetate. Arrows indicate positive MMP-1 staining. b Samples were stained with rabbit anti-MMP-3 and counterstained with 0.5% Methyl Green in 0.1 M Sodium Acetate. Arrows indicate positive MMP-3 staining. c Samples were stained with mouse anti-MMP-13 and nuclei were stained with DAPI. Arrows indicate positive MMP-13 staining. The percent area staining positive for MMP-13 in the 10 and 29 week old samples was calculated as the area with MMP-13 staining along the articular cartilage area against the total articular cartilage area. For experiments involving either 10 or 29 week old mice, the middle three metatarsal bones isolated from three animals were used. Each data point in the graphs represents an individual metatarsal bone. Each treatment was repeated 5–11 times. Negative controls using goat anti-rabbit IgG or biotinylated horse anti-mouse IgG followed by streptavidin DyLight 594 alone are shown in the Additional file 2 Figure S1. Data are plotted as mean ± SEM. * p = 0.0281 (Mann–Whitney test)
    Figure Legend Snippet: PEDF-deficiency protects against IL-1β-induced MMP-1, MMP-3 and MMP-13 protein production in metatarsal bone cultures. Metatarsal bones from 10 and 29 week old wild type or PEDF-deficient mice were harvested and cultured in the presence or absence of 10 ng/mL IL-1β for 7 days. Cryosectioned samples were analyzed for MMP protein levels by immunohistochemistry (IHC). AC: articular cartilage. SB: subchondral bone. Scale bar = 100 μm. a Samples were stained with rabbit anti-MMP-1 and counterstained with 0.5% Methyl Green in 0.1 M Sodium Acetate. Arrows indicate positive MMP-1 staining. b Samples were stained with rabbit anti-MMP-3 and counterstained with 0.5% Methyl Green in 0.1 M Sodium Acetate. Arrows indicate positive MMP-3 staining. c Samples were stained with mouse anti-MMP-13 and nuclei were stained with DAPI. Arrows indicate positive MMP-13 staining. The percent area staining positive for MMP-13 in the 10 and 29 week old samples was calculated as the area with MMP-13 staining along the articular cartilage area against the total articular cartilage area. For experiments involving either 10 or 29 week old mice, the middle three metatarsal bones isolated from three animals were used. Each data point in the graphs represents an individual metatarsal bone. Each treatment was repeated 5–11 times. Negative controls using goat anti-rabbit IgG or biotinylated horse anti-mouse IgG followed by streptavidin DyLight 594 alone are shown in the Additional file 2 Figure S1. Data are plotted as mean ± SEM. * p = 0.0281 (Mann–Whitney test)

    Techniques Used: Mouse Assay, Cell Culture, Immunohistochemistry, Staining, Isolation, MANN-WHITNEY

    PEDF expression is elevated in human osteoarthritis (OA) cartilage specimens. Normal samples were obtained from the National Disease Research Interchange and OA samples were obtained from patients undergoing total knee replacement surgery. a Normal sample stained with Safranin O and counterstained with Hematoxylin and Fast Green. Mankin score = 1. b Immunohistochemistry (IHC) analysis on a normal cartilage sample using a mouse anti-PEDF antibody and counterstained with Methyl Green. Magnified superficial (b') and deeper (b'') zones are shown from insets. c OA sample stained with Safranin O and counterstained with Hematoxylin and Fast Green. Mankin score = 5. d IHC analysis on an OA sample using a mouse anti-PEDF antibody and counterstained with Methyl Green. Magnified superficial (d') and deeper (d'') zones are shown from insets. Arrows indicate positive PEDF staining. A negative control using a biotinylated horse anti-mouse IgG secondary antibody alone is shown in the Additional file 2 : Figure S1. Scale bar = 200 μm
    Figure Legend Snippet: PEDF expression is elevated in human osteoarthritis (OA) cartilage specimens. Normal samples were obtained from the National Disease Research Interchange and OA samples were obtained from patients undergoing total knee replacement surgery. a Normal sample stained with Safranin O and counterstained with Hematoxylin and Fast Green. Mankin score = 1. b Immunohistochemistry (IHC) analysis on a normal cartilage sample using a mouse anti-PEDF antibody and counterstained with Methyl Green. Magnified superficial (b') and deeper (b'') zones are shown from insets. c OA sample stained with Safranin O and counterstained with Hematoxylin and Fast Green. Mankin score = 5. d IHC analysis on an OA sample using a mouse anti-PEDF antibody and counterstained with Methyl Green. Magnified superficial (d') and deeper (d'') zones are shown from insets. Arrows indicate positive PEDF staining. A negative control using a biotinylated horse anti-mouse IgG secondary antibody alone is shown in the Additional file 2 : Figure S1. Scale bar = 200 μm

    Techniques Used: Expressing, Staining, Immunohistochemistry, Negative Control

    26) Product Images from "UNG protects B cells from AID-induced telomere loss"

    Article Title: UNG protects B cells from AID-induced telomere loss

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20160635

    AID induces telomere loss in UNG-deficient B cells. (A) Possible outcomes after AID-dependent DNA deaminations are processed by UNG in B cells. (B, left) Illustration of typical FISH staining with a telomere-specific probe in metaphase chromosomes from normal cells and cells with STL. (Right) Effect of UNG inhibition via Ugi expression on the proportion of metaphases with STL in different CH12F3 lines expressing scrambled (scr) control or two different shRNAs that deplete AID, before and after stimulating for CSR to IgA. Post-stim., post-simulation. (C, left) Representative pictures of FISH on metaphase chromosomes in wild-type, Ung −/− , and Ung −/− Aicda −/− mouse splenic B cells stimulated for CSR to IgG1. Telomeres were hybridized with an Alexa Fluor 488–[TTAGGG] 4 probe (in green); total DNA was stained with DAPI (in blue). Arrowheads indicate missing telomere staining from single sister chromatids. Bars, 2 µm. (Right) Quantification of STL per metaphase after FISH analysis. Error bars represent mean + SD from at least three independent experiments. (D, left) Western analysis of wild-type AID or AIDE58A levels in CH12F3 Ugi cells. (Right) Quantification of metaphases with STL from CH12F3 Ugi cells expressing GFP, AID, or AIDE58A. (E, left) Illustration of CO-FISH staining. Leading-strand telomeres are shown in red, and lagging-strand telomeres are in green. (Middle) Representative pictures of CO-FISH in B cells at 4 d after stimulation with LPS and IL-4. Arrowheads indicate missing telomere staining from leading-strand telomeres. Bars, 2 µm. (Right) Quantification of STL per metaphase after CO-FISH analysis. (B, D, and E) Data show the mean + SD of three independent experiments, in which 50 metaphases per cell line were analyzed in each experiment.
    Figure Legend Snippet: AID induces telomere loss in UNG-deficient B cells. (A) Possible outcomes after AID-dependent DNA deaminations are processed by UNG in B cells. (B, left) Illustration of typical FISH staining with a telomere-specific probe in metaphase chromosomes from normal cells and cells with STL. (Right) Effect of UNG inhibition via Ugi expression on the proportion of metaphases with STL in different CH12F3 lines expressing scrambled (scr) control or two different shRNAs that deplete AID, before and after stimulating for CSR to IgA. Post-stim., post-simulation. (C, left) Representative pictures of FISH on metaphase chromosomes in wild-type, Ung −/− , and Ung −/− Aicda −/− mouse splenic B cells stimulated for CSR to IgG1. Telomeres were hybridized with an Alexa Fluor 488–[TTAGGG] 4 probe (in green); total DNA was stained with DAPI (in blue). Arrowheads indicate missing telomere staining from single sister chromatids. Bars, 2 µm. (Right) Quantification of STL per metaphase after FISH analysis. Error bars represent mean + SD from at least three independent experiments. (D, left) Western analysis of wild-type AID or AIDE58A levels in CH12F3 Ugi cells. (Right) Quantification of metaphases with STL from CH12F3 Ugi cells expressing GFP, AID, or AIDE58A. (E, left) Illustration of CO-FISH staining. Leading-strand telomeres are shown in red, and lagging-strand telomeres are in green. (Middle) Representative pictures of CO-FISH in B cells at 4 d after stimulation with LPS and IL-4. Arrowheads indicate missing telomere staining from leading-strand telomeres. Bars, 2 µm. (Right) Quantification of STL per metaphase after CO-FISH analysis. (B, D, and E) Data show the mean + SD of three independent experiments, in which 50 metaphases per cell line were analyzed in each experiment.

    Techniques Used: Fluorescence In Situ Hybridization, Staining, Inhibition, Expressing, Western Blot

    Mismatch repair factors mediate AID-induced STL in Ung-deficient B cells. (A) Possible outcomes of MSH2/MSH6-initiated repair of AID-induced DNA deaminations in B cells. (B) Western blot analysis of MSH2 in CH12F3 cells expressing the indicated shRNAs. scr, scrambled. (C) Quantification of the proportion of STL per 50 metaphases in each of the different CH12F3 lines expressing or not expressing Ugi and scrambled control or two different shRNAs that deplete MSH2, before and after stimulation of CSR to IgA. Error bars represent mean + SD from at least three independent experiments. (D, left) Representative ChIP performed with the indicated antibodies in wild-type, Ung −/− , and Ung −/− Aicda −/− splenic B cells stimulated for CSR to IgG1 and analyzed by dot blotting using telomeric or Alu probes. (Right) Plot of the mean + SD dot blot signals for the telomeric probe from three independent experiments. (E) UNG activity assay using a fluorescein-labeled oligonucleotide containing a single dU, incubated with cell extracts (10 µg protein) from the indicated CH12F3 lines used in C. Substrate and product, indicated on the left, were resolved on 15% TBE-urea polyacrylamide gels. Western blot of γ-tubulin level was used as a loading control. (F) Terminal restriction fragment analysis of TTAGGG repeats in stimulated CH12F3 and CH12F3-Ugi cells expressing the indicated shRNAs via Southern blotting in native or denatured conditions. sub-tel., subtelomeric. (G, left) Diagram showing the expected outcomes after treatment of genomic DNA with exonuclease I before the TRF analyses of TTAGGG repeats. The 3′ to 5′ single-strand exonuclease activity of ExoI will remove the telomeric 3′ G-rich overhang. Therefore, the signal for single-stranded TTAGGG repeats will be lost in a TRF analysis in native conditions. However, in telomeres with short gaps or nicks in the C-rich strand, ExoI activity will expose G-rich single-stranded gaps that can be detected in a TRF analysis in native conditions. (Right) Representative Southern blots of TRF after ExoI treatment in native and denatured conditions and quantification of telomeric ssDNA/dsDNA ratio in ExoI-treated genomic DNA. Error bars represent mean + SD from at least three independent experiments. *, P
    Figure Legend Snippet: Mismatch repair factors mediate AID-induced STL in Ung-deficient B cells. (A) Possible outcomes of MSH2/MSH6-initiated repair of AID-induced DNA deaminations in B cells. (B) Western blot analysis of MSH2 in CH12F3 cells expressing the indicated shRNAs. scr, scrambled. (C) Quantification of the proportion of STL per 50 metaphases in each of the different CH12F3 lines expressing or not expressing Ugi and scrambled control or two different shRNAs that deplete MSH2, before and after stimulation of CSR to IgA. Error bars represent mean + SD from at least three independent experiments. (D, left) Representative ChIP performed with the indicated antibodies in wild-type, Ung −/− , and Ung −/− Aicda −/− splenic B cells stimulated for CSR to IgG1 and analyzed by dot blotting using telomeric or Alu probes. (Right) Plot of the mean + SD dot blot signals for the telomeric probe from three independent experiments. (E) UNG activity assay using a fluorescein-labeled oligonucleotide containing a single dU, incubated with cell extracts (10 µg protein) from the indicated CH12F3 lines used in C. Substrate and product, indicated on the left, were resolved on 15% TBE-urea polyacrylamide gels. Western blot of γ-tubulin level was used as a loading control. (F) Terminal restriction fragment analysis of TTAGGG repeats in stimulated CH12F3 and CH12F3-Ugi cells expressing the indicated shRNAs via Southern blotting in native or denatured conditions. sub-tel., subtelomeric. (G, left) Diagram showing the expected outcomes after treatment of genomic DNA with exonuclease I before the TRF analyses of TTAGGG repeats. The 3′ to 5′ single-strand exonuclease activity of ExoI will remove the telomeric 3′ G-rich overhang. Therefore, the signal for single-stranded TTAGGG repeats will be lost in a TRF analysis in native conditions. However, in telomeres with short gaps or nicks in the C-rich strand, ExoI activity will expose G-rich single-stranded gaps that can be detected in a TRF analysis in native conditions. (Right) Representative Southern blots of TRF after ExoI treatment in native and denatured conditions and quantification of telomeric ssDNA/dsDNA ratio in ExoI-treated genomic DNA. Error bars represent mean + SD from at least three independent experiments. *, P

    Techniques Used: Western Blot, Expressing, Chromatin Immunoprecipitation, Dot Blot, Activity Assay, Labeling, Incubation, Southern Blot

    AID interacts with telomeres in B cells during CSR. (A) Schematic depiction of similarities between telomeres and S regions and location of AID’s preferred target sequences (WRCY). Note that WRCY motifs are present in both S-region strands but exclusively in the C-rich strand in telomeres. Sub-tel, subtelomeric. RPA, replication protein A; V, variable region. (B) Western blot analysis of AID expression in CH12F3 cells after cytokine stimulation for CSR. (C) Representative dot blot analysis of ChIP assays using anti-AID and IgG control in stimulated CH12F3 B cells. Dot blots with 5% of the input or the immunoprecipitates were analyzed via Southern blot with telomeric or Alu repeat probes. (D) Quantification of AID accumulation at telomeres (Telo) and Alu repeats by dot blot, as in C, as well as Sμ and Cμ regions of the Igh locus (by quantitative PCR [Q-PCR]) in CH12F3 cells stimulated for CSR, from at least three independent experiments. post-stim., post-stimulation. Error bars represent SD. (E, left) Western blot analysis of AID expression in CH12F3 cells expressing the indicated shRNAs. (Right) Representative ChIPs in CH12F3 B cells with the indicated antibodies out of three independent experiments. Coimmunoprecipitated telomeric DNA was detected via Southern blot with a telomeric (tel.) probe in dot blots. (F) One representative of three independent ChIP assays, as in C but in splenic B cells purified from Aicda +/+ or Aicda −/− mice, and stimulated with LPS and IL-4 for 72 h. ChIP for the telomeric (Tel) protein TRF1 was included as a positive control. (G) ChIPs in CH12F3 B cells with the indicated antibodies. (Right) Quantification of the dot blot signals after hybridization with a telomeric probe. (H) Northern blot with a telomeric probe showing the level of telomeric transcripts in wild-type splenic B cells before and after stimulation for CSR. EtBr, ethidium bromide. (Right) Quantification of Northern signals. (G and H) Data show mean + SD values obtained at each time point from three independent experiments.
    Figure Legend Snippet: AID interacts with telomeres in B cells during CSR. (A) Schematic depiction of similarities between telomeres and S regions and location of AID’s preferred target sequences (WRCY). Note that WRCY motifs are present in both S-region strands but exclusively in the C-rich strand in telomeres. Sub-tel, subtelomeric. RPA, replication protein A; V, variable region. (B) Western blot analysis of AID expression in CH12F3 cells after cytokine stimulation for CSR. (C) Representative dot blot analysis of ChIP assays using anti-AID and IgG control in stimulated CH12F3 B cells. Dot blots with 5% of the input or the immunoprecipitates were analyzed via Southern blot with telomeric or Alu repeat probes. (D) Quantification of AID accumulation at telomeres (Telo) and Alu repeats by dot blot, as in C, as well as Sμ and Cμ regions of the Igh locus (by quantitative PCR [Q-PCR]) in CH12F3 cells stimulated for CSR, from at least three independent experiments. post-stim., post-stimulation. Error bars represent SD. (E, left) Western blot analysis of AID expression in CH12F3 cells expressing the indicated shRNAs. (Right) Representative ChIPs in CH12F3 B cells with the indicated antibodies out of three independent experiments. Coimmunoprecipitated telomeric DNA was detected via Southern blot with a telomeric (tel.) probe in dot blots. (F) One representative of three independent ChIP assays, as in C but in splenic B cells purified from Aicda +/+ or Aicda −/− mice, and stimulated with LPS and IL-4 for 72 h. ChIP for the telomeric (Tel) protein TRF1 was included as a positive control. (G) ChIPs in CH12F3 B cells with the indicated antibodies. (Right) Quantification of the dot blot signals after hybridization with a telomeric probe. (H) Northern blot with a telomeric probe showing the level of telomeric transcripts in wild-type splenic B cells before and after stimulation for CSR. EtBr, ethidium bromide. (Right) Quantification of Northern signals. (G and H) Data show mean + SD values obtained at each time point from three independent experiments.

    Techniques Used: Recombinase Polymerase Amplification, Western Blot, Expressing, Dot Blot, Chromatin Immunoprecipitation, Southern Blot, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Purification, Mouse Assay, Positive Control, Hybridization, Northern Blot

    27) Product Images from "Altered Amyloid-β Metabolism and Deposition in Genomic-based β-Secretase Transgenic Mice *"

    Article Title: Altered Amyloid-β Metabolism and Deposition in Genomic-based β-Secretase Transgenic Mice *

    Journal: The Journal of biological chemistry

    doi: 10.1074/jbc.M409680200

    Human BACE1 immunohistochemical localization Brains from 2-month-old BACE1 transgenic ( B, D, F ) and nontransgenic ( A, C, E ) animals were cryoprotected in 30% sucrose after perfusion with 4% paraformaldehyde. Sagittal sections were analyzed by staining with polyclonal antibody B279 followed by staining with biotinylated anti-goat secondary antibody ( A - F ) or Alexa-488-conjugated secondary antibody ( G and H ). Positive immunoreactivity is present in the olfactory bulb ( A and B ), hippocampus ( C and D ), and frontal cortex ( E and F ). Scale bar in F and H = 200 μ m.
    Figure Legend Snippet: Human BACE1 immunohistochemical localization Brains from 2-month-old BACE1 transgenic ( B, D, F ) and nontransgenic ( A, C, E ) animals were cryoprotected in 30% sucrose after perfusion with 4% paraformaldehyde. Sagittal sections were analyzed by staining with polyclonal antibody B279 followed by staining with biotinylated anti-goat secondary antibody ( A - F ) or Alexa-488-conjugated secondary antibody ( G and H ). Positive immunoreactivity is present in the olfactory bulb ( A and B ), hippocampus ( C and D ), and frontal cortex ( E and F ). Scale bar in F and H = 200 μ m.

    Techniques Used: Immunohistochemistry, Transgenic Assay, Staining

    28) Product Images from "Human APOE genotype affects intraneuronal Aβ1–42 accumulation in a lentiviral gene transfer model"

    Article Title: Human APOE genotype affects intraneuronal Aβ1–42 accumulation in a lentiviral gene transfer model

    Journal: Human Molecular Genetics

    doi: 10.1093/hmg/ddt525

    Intracellular accumulation of lentiviral Aβ 1–42 is greatest in APOE4-TR mice at 2 weeks after lentivirus injection. ( A ) Representative image of a coronal brain section with DAB staining of biotinylated MOAB2 for lentiviral Aβ 1–42
    Figure Legend Snippet: Intracellular accumulation of lentiviral Aβ 1–42 is greatest in APOE4-TR mice at 2 weeks after lentivirus injection. ( A ) Representative image of a coronal brain section with DAB staining of biotinylated MOAB2 for lentiviral Aβ 1–42

    Techniques Used: Mouse Assay, Injection, Staining

    29) Product Images from "VPAC2 receptor expression in human normal and neoplastic tissues: evaluation of the novel MAB SP235"

    Article Title: VPAC2 receptor expression in human normal and neoplastic tissues: evaluation of the novel MAB SP235

    Journal: Endocrine Connections

    doi: 10.1530/EC-14-0051

    SP235 immunohistochemistry of human normal and neoplastic tissues. Sections were dewaxed, microwaved in citric acid and incubated with the rabbit monoclonal anti-VPAC2 antibody (SP235) at a dilution of 1:500. Sections were then sequentially treated with biotinylated anti-rabbit IgG and AB solution. Sections were then developed in AEC and lightly counterstained with haematoxylin. Insets in A and D, for adsorption controls the SP235 was incubated with 10 μg/ml of the peptide used for immunisations (+ peptide). Scale bar, A=B=C=F=I=250 μm and D=E=G=H=100 μm.
    Figure Legend Snippet: SP235 immunohistochemistry of human normal and neoplastic tissues. Sections were dewaxed, microwaved in citric acid and incubated with the rabbit monoclonal anti-VPAC2 antibody (SP235) at a dilution of 1:500. Sections were then sequentially treated with biotinylated anti-rabbit IgG and AB solution. Sections were then developed in AEC and lightly counterstained with haematoxylin. Insets in A and D, for adsorption controls the SP235 was incubated with 10 μg/ml of the peptide used for immunisations (+ peptide). Scale bar, A=B=C=F=I=250 μm and D=E=G=H=100 μm.

    Techniques Used: Immunohistochemistry, Incubation, Adsorption

    Characterisation of SP235 using mouse, rat and human tissues. (A) Comparision of the carboxyl-terminal sequences of mouse, rat and human VPAC2. The sequence depicted for the human VPAC2 was used for antibody generation. (B) Western blotting analysis of SP235 in various tissues. Tissue extracts from WT mice ( Vpac + / + ) and mice lacking Vpac2 ( Vpac2 −/− ) were separated on 7.5% SDS–polyacrylamide gels and blotted onto PVDF membranes. Membranes were then incubated with the rabbit monoclonal anti-VPAC2 antibody (SP235) at a dilution of 1:500. Membranes were developed using ECL. Ordinate, migration of protein molecular weight markers (kDa). (C) SP235 immunohistochemistry in rat and human tissues. Sections were dewaxed, microwaved in citric acid and incubated with the rabbit monoclonal anti-VPAC2 antibody (SP235) at a dilution of 1:500. Sections were then sequentially treated with biotinylated anti-rabbit IgG and AB solution. Sections were then developed in AEC and lightly counterstained with haematoxylin. Representative photomicrographs from one of five different tissue samples are shown. Scale bar: upper panel=200 μm and lower panel=100 μm.
    Figure Legend Snippet: Characterisation of SP235 using mouse, rat and human tissues. (A) Comparision of the carboxyl-terminal sequences of mouse, rat and human VPAC2. The sequence depicted for the human VPAC2 was used for antibody generation. (B) Western blotting analysis of SP235 in various tissues. Tissue extracts from WT mice ( Vpac + / + ) and mice lacking Vpac2 ( Vpac2 −/− ) were separated on 7.5% SDS–polyacrylamide gels and blotted onto PVDF membranes. Membranes were then incubated with the rabbit monoclonal anti-VPAC2 antibody (SP235) at a dilution of 1:500. Membranes were developed using ECL. Ordinate, migration of protein molecular weight markers (kDa). (C) SP235 immunohistochemistry in rat and human tissues. Sections were dewaxed, microwaved in citric acid and incubated with the rabbit monoclonal anti-VPAC2 antibody (SP235) at a dilution of 1:500. Sections were then sequentially treated with biotinylated anti-rabbit IgG and AB solution. Sections were then developed in AEC and lightly counterstained with haematoxylin. Representative photomicrographs from one of five different tissue samples are shown. Scale bar: upper panel=200 μm and lower panel=100 μm.

    Techniques Used: Sequencing, Western Blot, Mouse Assay, Incubation, Migration, Molecular Weight, Immunohistochemistry

    30) Product Images from "Human neural stem cells rapidly ameliorate symptomatic inflammation in early-stage ischemic-reperfusion cerebral injury"

    Article Title: Human neural stem cells rapidly ameliorate symptomatic inflammation in early-stage ischemic-reperfusion cerebral injury

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/scrt519

    Human NSC transplantation ameliorates BBB leakage. I. (A) Representative images of IgG staining in sham, MCAO/R and MCAO/R + hNSC-transplanted (Tx) mice showing BBB damage. Scale bar, 2 mm. (B) Quantification of IgG staining, expressed as a percentage of the volume showing IgG-positive staining per ipsilesional hemisphere (n = 5, *** P
    Figure Legend Snippet: Human NSC transplantation ameliorates BBB leakage. I. (A) Representative images of IgG staining in sham, MCAO/R and MCAO/R + hNSC-transplanted (Tx) mice showing BBB damage. Scale bar, 2 mm. (B) Quantification of IgG staining, expressed as a percentage of the volume showing IgG-positive staining per ipsilesional hemisphere (n = 5, *** P

    Techniques Used: Transplantation Assay, Staining, Mouse Assay

    31) Product Images from "Inflammation and Extracellular Matrix Degradation Mediated by Activated Transcription Factors Nuclear Factor-?B and Activator Protein-1 in Inflammatory Acne Lesions in Vivo"

    Article Title: Inflammation and Extracellular Matrix Degradation Mediated by Activated Transcription Factors Nuclear Factor-?B and Activator Protein-1 in Inflammatory Acne Lesions in Vivo

    Journal:

    doi:

    Activation of NF-κB in skin specimens from inflammatory acne lesions. An inflammatory papule ( B ) and adjacent clinically normal skin ( A ) were obtained from acne patients. A component of NF-κB, p65 protein, was detected with biotinylated
    Figure Legend Snippet: Activation of NF-κB in skin specimens from inflammatory acne lesions. An inflammatory papule ( B ) and adjacent clinically normal skin ( A ) were obtained from acne patients. A component of NF-κB, p65 protein, was detected with biotinylated

    Techniques Used: Activation Assay

    32) Product Images from "Expression and Functional Characterization of GABA Transporters in Crayfish Neurosecretory Cells"

    Article Title: Expression and Functional Characterization of GABA Transporters in Crayfish Neurosecretory Cells

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.22-21-09176.2002

    Immunoreactivity for plasma membrane transporters GAT-1, GAT-3, and GABA receptors in X-organ neurons in culture. Control culture cells were nonincubated with the primary antibodies, but they were processed with the avidin–biotin–peroxidase method and revealed with diaminobenzidine. Reactive cells were incubated overnight with the primary antibodies. The entire X-organ cells expressed immunoreactivity for GAT-1 and GAT-3 as well as the α1 subunit of the GABA receptor.
    Figure Legend Snippet: Immunoreactivity for plasma membrane transporters GAT-1, GAT-3, and GABA receptors in X-organ neurons in culture. Control culture cells were nonincubated with the primary antibodies, but they were processed with the avidin–biotin–peroxidase method and revealed with diaminobenzidine. Reactive cells were incubated overnight with the primary antibodies. The entire X-organ cells expressed immunoreactivity for GAT-1 and GAT-3 as well as the α1 subunit of the GABA receptor.

    Techniques Used: Avidin-Biotin Assay, Incubation

    33) Product Images from "Sarcospan-dependent Akt activation is required for utrophin expression and muscle regeneration"

    Article Title: Sarcospan-dependent Akt activation is required for utrophin expression and muscle regeneration

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201110032

    SSPN increases cell surface glycosylation in mdx muscle. (A and B) Transverse cryosections of quadriceps muscles from SSPN-Tg (A) or Akt transgenic (B) muscles were stained with biotinylated Wisteria floribunda agglutinin (WFA) and visualized by indirect immunofluorescence. Bars, 50 µm. (C) Skeletal muscle protein lysates from the indicated mouse models were enriched by WFA lectin affinity chromatography (WFA enrichment) and analyzed with indicated antibodies and overlayed (O/L) with WFA lectin (WFA O/L). (D) Skeletal muscle protein lysates were enriched by WFA lectin affinity chromatography (WFA enrichment) and subjected to the same analysis as described in C. (E) WFA and laminin overlay assays were performed on protein lysates enriched with succinylated WGA (sWGA) lectin chromatography from mdx and Akt transgenic mdx ( mdx Akt ) muscle. Mice were treated with doxycycline to induce Akt expression in skeletal muscle as described previously ( Peter et al., 2009 ). Laminin overlays (Lam O/L) represent binding to immobilized α-DG on nitrocellulose transfers. Immunoblotting with antibodies to α-DG is shown. (F) Levels of WFA binding to α-DG were quantitated by densitometry of bands from overlay assays, and data are expressed relative to mdx levels (100%). Error bars represent standard deviation of the mean ( n = 2–3 muscle preps per genotype). (G) Quantitative RT-PCR was used to investigate whether overexpression of SSPN alters RNA levels of CT GalNAc transferase ( Galgt2 ). RNA was isolated from WT, WT 1.5 , mdx , and mdx 1.5 skeletal muscle. Data are expressed relative to non-Tg WT controls. Error bars represent standard error of the mean (*, P
    Figure Legend Snippet: SSPN increases cell surface glycosylation in mdx muscle. (A and B) Transverse cryosections of quadriceps muscles from SSPN-Tg (A) or Akt transgenic (B) muscles were stained with biotinylated Wisteria floribunda agglutinin (WFA) and visualized by indirect immunofluorescence. Bars, 50 µm. (C) Skeletal muscle protein lysates from the indicated mouse models were enriched by WFA lectin affinity chromatography (WFA enrichment) and analyzed with indicated antibodies and overlayed (O/L) with WFA lectin (WFA O/L). (D) Skeletal muscle protein lysates were enriched by WFA lectin affinity chromatography (WFA enrichment) and subjected to the same analysis as described in C. (E) WFA and laminin overlay assays were performed on protein lysates enriched with succinylated WGA (sWGA) lectin chromatography from mdx and Akt transgenic mdx ( mdx Akt ) muscle. Mice were treated with doxycycline to induce Akt expression in skeletal muscle as described previously ( Peter et al., 2009 ). Laminin overlays (Lam O/L) represent binding to immobilized α-DG on nitrocellulose transfers. Immunoblotting with antibodies to α-DG is shown. (F) Levels of WFA binding to α-DG were quantitated by densitometry of bands from overlay assays, and data are expressed relative to mdx levels (100%). Error bars represent standard deviation of the mean ( n = 2–3 muscle preps per genotype). (G) Quantitative RT-PCR was used to investigate whether overexpression of SSPN alters RNA levels of CT GalNAc transferase ( Galgt2 ). RNA was isolated from WT, WT 1.5 , mdx , and mdx 1.5 skeletal muscle. Data are expressed relative to non-Tg WT controls. Error bars represent standard error of the mean (*, P

    Techniques Used: Transgenic Assay, Staining, Immunofluorescence, Affinity Chromatography, Whole Genome Amplification, Chromatography, Mouse Assay, Expressing, Laser Capture Microdissection, Binding Assay, Standard Deviation, Quantitative RT-PCR, Over Expression, Isolation

    SSPN regulates utrophin levels and glycosylation of α-DG in myd mice. (A) Transverse cryosections of quadriceps muscles were stained with the indicated antibodies and overlayed with biotinylated WFA. (B) Transverse cryosections of quadriceps muscles were stained with indicated antibodies and overlayed with biotinylated WFA. Staining with IIH6 antibodies, which recognize LARGE epitopes on α-DG, was not detected in myd samples, as expected ( Fig. S4 ). Transverse cryosections of skeletal muscle from 4–6-wk-old myd , SSPN-Tg: myd ( myd 3.0 ), and SSPN-deficient myd ( myd :SSPN −/− ) mice were stained with H E. Muscle sections were stained with antibodies to embryonic myosin heavy chain (eMHC; green) as a marker for newly regenerated myofibers. Mice were injected with Evans blue dye (EBD), a marker for membrane instability (visualized by red fluorescence). Sections were costained with laminin antibodies (green fluorescence) to visualize individual fibers. (C) Quantification of central nucleation, Evans blue dye–positive fibers, and eMHC-positive fibers is expressed as a percentage of total fibers. Error bars represent standard deviation of the mean ( n = 4 quadriceps per genotype). (D–G) Skeletal muscles from the indicated mice were enriched using either sWGA or WFA lectin chromatography. Immunoblots of 10-µg protein eluates are shown. A.U., arbitrary unit; Utr, utrophin. Bars, 50 µm.
    Figure Legend Snippet: SSPN regulates utrophin levels and glycosylation of α-DG in myd mice. (A) Transverse cryosections of quadriceps muscles were stained with the indicated antibodies and overlayed with biotinylated WFA. (B) Transverse cryosections of quadriceps muscles were stained with indicated antibodies and overlayed with biotinylated WFA. Staining with IIH6 antibodies, which recognize LARGE epitopes on α-DG, was not detected in myd samples, as expected ( Fig. S4 ). Transverse cryosections of skeletal muscle from 4–6-wk-old myd , SSPN-Tg: myd ( myd 3.0 ), and SSPN-deficient myd ( myd :SSPN −/− ) mice were stained with H E. Muscle sections were stained with antibodies to embryonic myosin heavy chain (eMHC; green) as a marker for newly regenerated myofibers. Mice were injected with Evans blue dye (EBD), a marker for membrane instability (visualized by red fluorescence). Sections were costained with laminin antibodies (green fluorescence) to visualize individual fibers. (C) Quantification of central nucleation, Evans blue dye–positive fibers, and eMHC-positive fibers is expressed as a percentage of total fibers. Error bars represent standard deviation of the mean ( n = 4 quadriceps per genotype). (D–G) Skeletal muscles from the indicated mice were enriched using either sWGA or WFA lectin chromatography. Immunoblots of 10-µg protein eluates are shown. A.U., arbitrary unit; Utr, utrophin. Bars, 50 µm.

    Techniques Used: Mouse Assay, Staining, Marker, Injection, Fluorescence, Standard Deviation, Chromatography, Western Blot

    34) Product Images from "Liver X Receptor Activation Enhances Cholesterol Loss from the Brain, Decreases Neuroinflammation, and Increases Survival of the NPC1 Mouse"

    Article Title: Liver X Receptor Activation Enhances Cholesterol Loss from the Brain, Decreases Neuroinflammation, and Increases Survival of the NPC1 Mouse

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.4823-07.2007

    Cholesterol accumulation in cerebellum and mRNA levels for proteins involved in cholesterol metabolism. A–C , The cerebellum was removed from npc1 +/+ mice 49 d of age that had been given the low-cholesterol diet since weaning and from npc1 −/− animals given this diet alone or with T1317. Frozen sections of this region were stained with biotinylated perfringolysin peptide (BCθ) to identify sequestration of unesterified cholesterol in the cells of the npc1 −/− animals. m and g identify the molecular and granular layers, respectively, whereas pc represents Purkinje cells. D , Relative mRNA levels for proteins potentially involved in the cellular trafficking of cholesterol ( n = 6). E , Relative mRNA levels for proteins potentially involved in the synthesis or degradation of cholesterol ( n = 6).
    Figure Legend Snippet: Cholesterol accumulation in cerebellum and mRNA levels for proteins involved in cholesterol metabolism. A–C , The cerebellum was removed from npc1 +/+ mice 49 d of age that had been given the low-cholesterol diet since weaning and from npc1 −/− animals given this diet alone or with T1317. Frozen sections of this region were stained with biotinylated perfringolysin peptide (BCθ) to identify sequestration of unesterified cholesterol in the cells of the npc1 −/− animals. m and g identify the molecular and granular layers, respectively, whereas pc represents Purkinje cells. D , Relative mRNA levels for proteins potentially involved in the cellular trafficking of cholesterol ( n = 6). E , Relative mRNA levels for proteins potentially involved in the synthesis or degradation of cholesterol ( n = 6).

    Techniques Used: Mouse Assay, Staining

    35) Product Images from "Mechanism and treatment for the learning and memory deficits associated with mouse models of Noonan syndrome"

    Article Title: Mechanism and treatment for the learning and memory deficits associated with mouse models of Noonan syndrome

    Journal: Nature neuroscience

    doi: 10.1038/nn.3863

    PTPN11 D61G overexpression increases surface AMPA receptor expression a. and b. Representative images of surface GluA1 staining in cultured neurons. GFP alone (a) or PTPN11 D61G and GFP (b) were co–expressed using a bicistronic Sindbis viral vector in cultured hippocampal neurons (DIV21). Scale, 20μm. c. Representative images of western blotting of total and biotinylated surface proteins. Cadherin and Rab-4 were used as markers for surface and cytosol expression, respectively. Full-length blots/gels are presented in Supplementary Figure 11 . d. Surface expression of GluA1 was significantly increased in PTPN11 D61G expressing neurons compared to WT PTPN11 expressing neurons, while the total expression level of GluA1 did not differ between WT PTPN11 and PTPN11 D61G transfected neurons.
    Figure Legend Snippet: PTPN11 D61G overexpression increases surface AMPA receptor expression a. and b. Representative images of surface GluA1 staining in cultured neurons. GFP alone (a) or PTPN11 D61G and GFP (b) were co–expressed using a bicistronic Sindbis viral vector in cultured hippocampal neurons (DIV21). Scale, 20μm. c. Representative images of western blotting of total and biotinylated surface proteins. Cadherin and Rab-4 were used as markers for surface and cytosol expression, respectively. Full-length blots/gels are presented in Supplementary Figure 11 . d. Surface expression of GluA1 was significantly increased in PTPN11 D61G expressing neurons compared to WT PTPN11 expressing neurons, while the total expression level of GluA1 did not differ between WT PTPN11 and PTPN11 D61G transfected neurons.

    Techniques Used: Over Expression, Expressing, Staining, Cell Culture, Plasmid Preparation, Western Blot, Transfection

    36) Product Images from "Antibodies to CD20 and MHC class II antigen bound to B-lymphoma cells accumulate in shed cytoplasmic fragments"

    Article Title: Antibodies to CD20 and MHC class II antigen bound to B-lymphoma cells accumulate in shed cytoplasmic fragments

    Journal: British Journal of Cancer

    doi: 10.1038/sj.bjc.6602131

    Immunoperoxidase staining of B-lymphoma cells after overnight incubation with anti-CD20 (1F5). Cells were deposited on cytocentrifuge slides, fixed with formaldehyde, permeabilised with saponin, and stained with a biotinylated horse anti-mouse IgG, followed by a complex of streptavidin and peroxidase. Observation was with an × 40 objective, except as noted. ( A ) Raji cells, showing prominent staining of JN spots. ( B ) RL cells, showing dark staining of apparently extracellular objects, referred to as CFs. ( C ) A lower-power photograph of RL cells (× 10), to show the general staining pattern. ( D ) RL cells stained in the absence of saponin, to show antigen that is accessible without permeabilisation. Control Abs of the same subclass produced no brown staining.
    Figure Legend Snippet: Immunoperoxidase staining of B-lymphoma cells after overnight incubation with anti-CD20 (1F5). Cells were deposited on cytocentrifuge slides, fixed with formaldehyde, permeabilised with saponin, and stained with a biotinylated horse anti-mouse IgG, followed by a complex of streptavidin and peroxidase. Observation was with an × 40 objective, except as noted. ( A ) Raji cells, showing prominent staining of JN spots. ( B ) RL cells, showing dark staining of apparently extracellular objects, referred to as CFs. ( C ) A lower-power photograph of RL cells (× 10), to show the general staining pattern. ( D ) RL cells stained in the absence of saponin, to show antigen that is accessible without permeabilisation. Control Abs of the same subclass produced no brown staining.

    Techniques Used: Immunoperoxidase Staining, Incubation, Staining, Produced

    37) Product Images from "Myocardial protection from ischemia/reperfusion injury by endogenous and exogenous HGF"

    Article Title: Myocardial protection from ischemia/reperfusion injury by endogenous and exogenous HGF

    Journal: Journal of Clinical Investigation

    doi:

    Adverse effects of neutralization of endogenous HGF on the ischemia/reperfusion injury model. ( a ) Specificity of the neutralizing antibody to HGF. Plasma from a rat with ischemia/reperfusion injury was immunoprecipitated with normal IgG (lane 1) or anti–rat HGF IgG (lane 2), and immunoreactive proteins were detected by Western blot, using biotinylated anti–rat HGF IgG. ( b ) Immunohistochemical staining of infarcted hearts with α-sarcomeric actin to depict the infarct area and its quantification. Anti–rat HGF IgG ( n = 10) or normal IgG ( n = 10) was injected 20 minutes before coronary occlusion, and every 12 hours after reperfusion. Forty-eight hours after operation, rats were killed and histological and biochemical analyses were made. Arrowheads indicate the α-sarcomeric actin–negative infarct area (original magnification, ×40). A P
    Figure Legend Snippet: Adverse effects of neutralization of endogenous HGF on the ischemia/reperfusion injury model. ( a ) Specificity of the neutralizing antibody to HGF. Plasma from a rat with ischemia/reperfusion injury was immunoprecipitated with normal IgG (lane 1) or anti–rat HGF IgG (lane 2), and immunoreactive proteins were detected by Western blot, using biotinylated anti–rat HGF IgG. ( b ) Immunohistochemical staining of infarcted hearts with α-sarcomeric actin to depict the infarct area and its quantification. Anti–rat HGF IgG ( n = 10) or normal IgG ( n = 10) was injected 20 minutes before coronary occlusion, and every 12 hours after reperfusion. Forty-eight hours after operation, rats were killed and histological and biochemical analyses were made. Arrowheads indicate the α-sarcomeric actin–negative infarct area (original magnification, ×40). A P

    Techniques Used: Neutralization, Immunoprecipitation, Western Blot, Immunohistochemistry, Staining, Injection

    38) Product Images from "Identification of a Novel UT-B Urea Transporter in Human Urothelial Cancer"

    Article Title: Identification of a Novel UT-B Urea Transporter in Human Urothelial Cancer

    Journal: Frontiers in Physiology

    doi: 10.3389/fphys.2017.00245

    Immunohistochemical analysis of UT-B expression with bladder cancer tissues . Human bladder cancer tissue arrays paired with normal tissues were processed for immunostaining with UT-B antibody (1:400), followed by biotinylated anti-rabbit antibody, and ABC (Vector laboratories). Color was developed by DAB. The nuclei were counterstained with hematoxylin. Representative pictures of UT-B staining in bladder cancer are shown from total 63 cases ( n = 73) (magnification: x200).
    Figure Legend Snippet: Immunohistochemical analysis of UT-B expression with bladder cancer tissues . Human bladder cancer tissue arrays paired with normal tissues were processed for immunostaining with UT-B antibody (1:400), followed by biotinylated anti-rabbit antibody, and ABC (Vector laboratories). Color was developed by DAB. The nuclei were counterstained with hematoxylin. Representative pictures of UT-B staining in bladder cancer are shown from total 63 cases ( n = 73) (magnification: x200).

    Techniques Used: Immunohistochemistry, Expressing, Immunostaining, Plasmid Preparation, Staining

    39) Product Images from "Establishment and characterization of a telomerase immortalized human gingival epithelial cell line"

    Article Title: Establishment and characterization of a telomerase immortalized human gingival epithelial cell line

    Journal: Journal of periodontal research

    doi: 10.1111/jre.12059

    Immunohistochemical detection of cytokeratins in TIGKs TIGKs were cultured in K-SFM medium for 19 days to allow cells within the central regions of colonies to form stratified layers 2-3 cells thick. Cells were probed with rabbit polyclonal IgG to cytokeratin 14 (A) and with mouse monoclonal antibodies to cytokeratin 19 (B), cytokeratin 13 (C), cytokeratin 16 (D) and cytokeratin 10 (E). Non-immune rabbit (F) and mouse (G) IgGs served as negative controls. Immunodetection was performed using the avidin-biotin-peroxidase complex. Cells were imaged under differential interference contrast optics without counterstaining. Bar in lower right of panel E = 100 μm for all panels.
    Figure Legend Snippet: Immunohistochemical detection of cytokeratins in TIGKs TIGKs were cultured in K-SFM medium for 19 days to allow cells within the central regions of colonies to form stratified layers 2-3 cells thick. Cells were probed with rabbit polyclonal IgG to cytokeratin 14 (A) and with mouse monoclonal antibodies to cytokeratin 19 (B), cytokeratin 13 (C), cytokeratin 16 (D) and cytokeratin 10 (E). Non-immune rabbit (F) and mouse (G) IgGs served as negative controls. Immunodetection was performed using the avidin-biotin-peroxidase complex. Cells were imaged under differential interference contrast optics without counterstaining. Bar in lower right of panel E = 100 μm for all panels.

    Techniques Used: Immunohistochemistry, Cell Culture, Immunodetection, Avidin-Biotin Assay

    40) Product Images from "Fas and Fas Ligand Are Up-Regulated in Pulmonary Edema Fluid and Lung Tissue of Patients with Acute Lung Injury and the Acute Respiratory Distress Syndrome"

    Article Title: Fas and Fas Ligand Are Up-Regulated in Pulmonary Edema Fluid and Lung Tissue of Patients with Acute Lung Injury and the Acute Respiratory Distress Syndrome

    Journal: The American Journal of Pathology

    doi:

    Localization of markers of apoptosis in lung tissue sections from patients. The left column shows lung tissue sections from a patient who died with ALI or ARDS [identified as “(+) ARDS”]. The right column shows lung tissue sections from a patient who died without pulmonary disease [identified as “(−) ARDS”]. a to f: Tissue sections that are counterstained with hematoxylin. g to j: Tissue sections that were imaged using differential interference contrast optics, without counterstain, because the epithelial cell immunostain reaction product was subtle. The rows of pictures are matched for one marker of apoptosis: TUNEL ( a and b ), caspase-3 ( c and d ), Bax ( e and f ), Bcl II ( g and h ), and p53 ( i and j ). Cells lining, and in, the alveolar walls demonstrate more TUNEL-labeled nuclei ( arrow ), caspase-3-labeled cytoplasm ( arrow ), Bax-labeled cytoplasm ( arrow ), and p53-labeled cytoplasm ( arrow ) in the tissue sections from the patients who died with ALI or ARDS compared to the patient who died without pulmonary disease. On the other hand, Bcl II-labeled cells lining, and in, the alveolar walls are more prominent in the tissue sections from the patient who died without pulmonary disease compared to the patient who died with ALI or ARDS, as expected. All of the panels are the same magnification.
    Figure Legend Snippet: Localization of markers of apoptosis in lung tissue sections from patients. The left column shows lung tissue sections from a patient who died with ALI or ARDS [identified as “(+) ARDS”]. The right column shows lung tissue sections from a patient who died without pulmonary disease [identified as “(−) ARDS”]. a to f: Tissue sections that are counterstained with hematoxylin. g to j: Tissue sections that were imaged using differential interference contrast optics, without counterstain, because the epithelial cell immunostain reaction product was subtle. The rows of pictures are matched for one marker of apoptosis: TUNEL ( a and b ), caspase-3 ( c and d ), Bax ( e and f ), Bcl II ( g and h ), and p53 ( i and j ). Cells lining, and in, the alveolar walls demonstrate more TUNEL-labeled nuclei ( arrow ), caspase-3-labeled cytoplasm ( arrow ), Bax-labeled cytoplasm ( arrow ), and p53-labeled cytoplasm ( arrow ) in the tissue sections from the patients who died with ALI or ARDS compared to the patient who died without pulmonary disease. On the other hand, Bcl II-labeled cells lining, and in, the alveolar walls are more prominent in the tissue sections from the patient who died without pulmonary disease compared to the patient who died with ALI or ARDS, as expected. All of the panels are the same magnification.

    Techniques Used: Marker, TUNEL Assay, Labeling

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    Incubation:

    Article Title: Combinational therapy using hypothermia and the immunophilin ligand FK506 to target altered pial arteriolar reactivity, axonal damage, and blood-brain barrier dysfunction after traumatic brain injury in rat
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    other:

    Article Title: Vasohibin as an endothelium-derived negative feedback regulator of angiogenesis
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    Next-Generation Sequencing:

    Article Title: Fluorescent Arc/Arg3.1 indicator mice: a versatile tool to study brain activity changes in vitro and in vivo
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    Article Title: Enhanced biocompatibility of CD47-functionalized vascular stents
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    Vector Laboratories biotinylated anti rabbit antibody
    Increased d4EGFP expression in a mouse model of Alzheimer’s disease Representative images of DG hippocampus (coronal sections) from one month-old Tg Arc/Arg3.1-d4EGFP and Tg 5XFAD /Tg Arc/Arg3.1-d4EGFP littermate mice are shown after visualization of d4EGFP by DAB (A, B) or immunofluorescence (C,D). Note the increased number of and labeling intensity of d4EGFP-positive neurons in CA1 (B) and DG granule cells (B, D) in the Tg 5XFAD /Tg Arc/Arg3.1-d4EGFP mice. Brain sections stained with anti-GFP antibody and visualized by <t>biotinylated</t> (DAB method) or FITC-conjugated secondary antibodies. The fluorescent images are single confocal scans. Abbreviations: db – dorsal blade of DG, vb – ventral blade of DG; scale bars = 250 μm (A,B) and 100 μm (C,D). (E) Count of d4EGFP-positive neurons in Tg 5XFAD /Tg Arc/Arg3.1-d4EGFP mice, as percent change from control Tg Arc/Arg3.1-d4EGFP littermates, in the DG: (% mean ± SD): 216.3 ± 7.7 vs. 100 ± 6.4 (**p
    Biotinylated Anti Rabbit Antibody, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Increased d4EGFP expression in a mouse model of Alzheimer’s disease Representative images of DG hippocampus (coronal sections) from one month-old Tg Arc/Arg3.1-d4EGFP and Tg 5XFAD /Tg Arc/Arg3.1-d4EGFP littermate mice are shown after visualization of d4EGFP by DAB (A, B) or immunofluorescence (C,D). Note the increased number of and labeling intensity of d4EGFP-positive neurons in CA1 (B) and DG granule cells (B, D) in the Tg 5XFAD /Tg Arc/Arg3.1-d4EGFP mice. Brain sections stained with anti-GFP antibody and visualized by biotinylated (DAB method) or FITC-conjugated secondary antibodies. The fluorescent images are single confocal scans. Abbreviations: db – dorsal blade of DG, vb – ventral blade of DG; scale bars = 250 μm (A,B) and 100 μm (C,D). (E) Count of d4EGFP-positive neurons in Tg 5XFAD /Tg Arc/Arg3.1-d4EGFP mice, as percent change from control Tg Arc/Arg3.1-d4EGFP littermates, in the DG: (% mean ± SD): 216.3 ± 7.7 vs. 100 ± 6.4 (**p

    Journal: Journal of neuroscience methods

    Article Title: Fluorescent Arc/Arg3.1 indicator mice: a versatile tool to study brain activity changes in vitro and in vivo

    doi: 10.1016/j.jneumeth.2009.07.015

    Figure Lengend Snippet: Increased d4EGFP expression in a mouse model of Alzheimer’s disease Representative images of DG hippocampus (coronal sections) from one month-old Tg Arc/Arg3.1-d4EGFP and Tg 5XFAD /Tg Arc/Arg3.1-d4EGFP littermate mice are shown after visualization of d4EGFP by DAB (A, B) or immunofluorescence (C,D). Note the increased number of and labeling intensity of d4EGFP-positive neurons in CA1 (B) and DG granule cells (B, D) in the Tg 5XFAD /Tg Arc/Arg3.1-d4EGFP mice. Brain sections stained with anti-GFP antibody and visualized by biotinylated (DAB method) or FITC-conjugated secondary antibodies. The fluorescent images are single confocal scans. Abbreviations: db – dorsal blade of DG, vb – ventral blade of DG; scale bars = 250 μm (A,B) and 100 μm (C,D). (E) Count of d4EGFP-positive neurons in Tg 5XFAD /Tg Arc/Arg3.1-d4EGFP mice, as percent change from control Tg Arc/Arg3.1-d4EGFP littermates, in the DG: (% mean ± SD): 216.3 ± 7.7 vs. 100 ± 6.4 (**p

    Article Snippet: Briefly, slices were blocked with 10% NGS containing 0.1% Triton X-100 antibody in PBS for 1 hr and reacted with either anti-Arc (Arg3.1 antiserum, provided by Dietmar Kuhl, dilution 1:1,500) or anti-GFP (Molecular Probes, Cat# , dilution 1:2,000) rabbit antibody in 1% NGS containing PBS overnight at 4 C. After reaction with biotinylated anti-rabbit antibody (Vector laboratory, dilution 1:200), immunoreactions were visualized with the ABC elite kit (Vector laboratory) and DAB development procedure.

    Techniques: Expressing, Mouse Assay, Immunofluorescence, Labeling, Staining

    KIAA1036 is preferentially expressed in ECs. ( A ) Expression of KIAA1036 in cultured cells. Cells were preincubated in 0.1% FCS/α-MEM for 12 hours and then stimulated with growth factors as follows: HUVECs with VEGF (1 nM), HASMCs with PDGF (1 nM), human fibroblasts with FGF-2 (2 nM), and keratinocytes with EGF (1 nM). Thereafter, total RNA was obtained and Northern blotting for vasohibin was performed. ( B ) Expression of KIAA1036 in vivo was examined by multiple-tissue Northern blot. ( C ) Localization of KIAA1036 protein in the placenta. Sections of human placenta were subjected to immunostaining. Anti_human CD31 mAb, anti_KIAA1036 mAb, or mouse IgG was used as the primary Ab. Scale bars: 100 μm. ( D ) Expression of KIAA1036 in human embryo. Northern blotting for vasohibin was performed using a human developmental total RNA Northern blot.

    Journal: Journal of Clinical Investigation

    Article Title: Vasohibin as an endothelium-derived negative feedback regulator of angiogenesis

    doi: 10.1172/JCI200421152

    Figure Lengend Snippet: KIAA1036 is preferentially expressed in ECs. ( A ) Expression of KIAA1036 in cultured cells. Cells were preincubated in 0.1% FCS/α-MEM for 12 hours and then stimulated with growth factors as follows: HUVECs with VEGF (1 nM), HASMCs with PDGF (1 nM), human fibroblasts with FGF-2 (2 nM), and keratinocytes with EGF (1 nM). Thereafter, total RNA was obtained and Northern blotting for vasohibin was performed. ( B ) Expression of KIAA1036 in vivo was examined by multiple-tissue Northern blot. ( C ) Localization of KIAA1036 protein in the placenta. Sections of human placenta were subjected to immunostaining. Anti_human CD31 mAb, anti_KIAA1036 mAb, or mouse IgG was used as the primary Ab. Scale bars: 100 μm. ( D ) Expression of KIAA1036 in human embryo. Northern blotting for vasohibin was performed using a human developmental total RNA Northern blot.

    Article Snippet: The materials used and their sources were as follows: VEGF165 and HGF (Sigma-Aldrich); FGF-2 and growth factor-reduced Matrigel (Collaborative Research Inc.); PlGF and TNF-α (R & D Systems Inc.); α-MEM, DMEM, and medium 199 (M199; Nissui Pharmaceutical Co.); endothelial basal medium (EBM) containing EC growth supplements (Clonetics Corp.); FBS (JRH Biosciences Inc.); Opti-MEM, Lipofectin, and Oligofectamine (Invitrogen Corp.); ISOGEN (Nippon Gene Co.); Hybond N+ membrane, nitrocellulose membranes, and protein A Sepharose (Amersham Biosciences); mouse anti–human CD31 mAb (DakoCytomation); rat anti–mouse CD31 Ab (Research Diagnostics Inc.); and biotin-conjugated anti–rabbit IgG (Vector Laboratories Inc.).

    Techniques: Expressing, Cell Culture, Northern Blot, In Vivo, Immunostaining

    CD47 Quantification. (A) The modification chemistry summarized in was utilized to facilitate quantification. Following PEI-PDT addition, primary amines were acetylated using sulfo-NHS-Acetate. recCD47 and pepCD47 were biotinylated using Sulfo-NHS-LC-LC-Biotin.

    Journal: Biomaterials

    Article Title: Enhanced biocompatibility of CD47-functionalized vascular stents

    doi: 10.1016/j.biomaterials.2016.02.008

    Figure Lengend Snippet: CD47 Quantification. (A) The modification chemistry summarized in was utilized to facilitate quantification. Following PEI-PDT addition, primary amines were acetylated using sulfo-NHS-Acetate. recCD47 and pepCD47 were biotinylated using Sulfo-NHS-LC-LC-Biotin.

    Article Snippet: The absolute values of CD47 immobilization density were obtained by plotting the OD data generated with the sets of foil samples to a standard OD curve obtained with escalating amounts of biotinylated goat anti-rabbit antibody (Vector Lab), disclosed to contain an average of 10 biotin groups per a 150 kDa molecule.

    Techniques: Modification

    Bar graph shows a comparison of the mean area of IgG immunostaining among groups. It must be noted that neither hypothermic intervention nor FK506 administration prevented the spatial extravasation of endogenous IgG. In addition, even the combination

    Journal: Journal of Cerebral Blood Flow & Metabolism

    Article Title: Combinational therapy using hypothermia and the immunophilin ligand FK506 to target altered pial arteriolar reactivity, axonal damage, and blood-brain barrier dysfunction after traumatic brain injury in rat

    doi: 10.1038/jcbfm.2010.208

    Figure Lengend Snippet: Bar graph shows a comparison of the mean area of IgG immunostaining among groups. It must be noted that neither hypothermic intervention nor FK506 administration prevented the spatial extravasation of endogenous IgG. In addition, even the combination

    Article Snippet: Next, the sections were incubated for 1 hour with biotinylated goat anti-rabbit immunoglobulin G (IgG) (Vector Laboratories Inc., Burlingame, CA, USA) diluted 1:1,000 in 1% normal goat serum in PBS.

    Techniques: Immunostaining