vector blue alkaline phosphatase substrate kit iii  (Vector Laboratories)


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    BCIP NBT Alkaline Phosphatase AP Substrate Kit 5 bromo 4 chloro 3 indolyl phosphate nitroblue tetrazolium
    Description:
    BCIP NBT Alkaline Phosphatase Substrate Kit has greater sensitivity than conventional substratesConsistent and reliableIdeal for IHC ICC ISH and blotsFluorescentHeat StablePermanent mountingIdeal for single and multiple labelingOne year expiry dateStock solutions supplied in convenient dropper bottles promoting ease of handlingNo wait times for mixing and dissolving powders or tabletsSufficient reagents to produce 200 ml of working solutionExcitation 645 685 nm Emission peaks at 823 and 855 nm Biotechniques 2007 42 756 759 and BioTechniques 2014 57 254 256 BCIP NBT AP Substrate Kit forms an indigo heat stable reaction product that can be dehydrated cleared and permanently mounted This chromogen can be used singly or in combination with other alkaline phosphatase or peroxidase substrates for multiple label applications This substrate can also be used for developing blots or for in situ hybridization At 20 30 minute development times the sensitivity of this substrate is equivalent to the other alkaline phosphatase substrates However BCIP NBT will continue to develop from several hours to overnight making it one of the most sensitive chromogenic substrates This kit contains stock solutions in convenient dropper bottles
    Catalog Number:
    SK-5400
    Price:
    None
    Category:
    Protein chromogenic detection reagents or kits or substrates
    Size:
    1 Kit
    Buy from Supplier


    Structured Review

    Vector Laboratories vector blue alkaline phosphatase substrate kit iii
    BCIP NBT Alkaline Phosphatase AP Substrate Kit 5 bromo 4 chloro 3 indolyl phosphate nitroblue tetrazolium
    BCIP NBT Alkaline Phosphatase Substrate Kit has greater sensitivity than conventional substratesConsistent and reliableIdeal for IHC ICC ISH and blotsFluorescentHeat StablePermanent mountingIdeal for single and multiple labelingOne year expiry dateStock solutions supplied in convenient dropper bottles promoting ease of handlingNo wait times for mixing and dissolving powders or tabletsSufficient reagents to produce 200 ml of working solutionExcitation 645 685 nm Emission peaks at 823 and 855 nm Biotechniques 2007 42 756 759 and BioTechniques 2014 57 254 256 BCIP NBT AP Substrate Kit forms an indigo heat stable reaction product that can be dehydrated cleared and permanently mounted This chromogen can be used singly or in combination with other alkaline phosphatase or peroxidase substrates for multiple label applications This substrate can also be used for developing blots or for in situ hybridization At 20 30 minute development times the sensitivity of this substrate is equivalent to the other alkaline phosphatase substrates However BCIP NBT will continue to develop from several hours to overnight making it one of the most sensitive chromogenic substrates This kit contains stock solutions in convenient dropper bottles
    https://www.bioz.com/result/vector blue alkaline phosphatase substrate kit iii/product/Vector Laboratories
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    vector blue alkaline phosphatase substrate kit iii - by Bioz Stars, 2021-04
    95/100 stars

    Images

    1) Product Images from "Heme–hemopexin complex attenuates neuronal cell death and stroke damage"

    Article Title: Heme–hemopexin complex attenuates neuronal cell death and stroke damage

    Journal: Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism

    doi: 10.1038/jcbfm.2009.19

    Heme–HPX induces HO1 expression in primary cortical neurons. Embryonic cortical neurons were incubated for 6 h with HHPX. HO1 induction was measured by RT-PCR ( A ) to analyze the relative level of HO1 mRNA or by Western blot ( B ) to determine HO1 protein, both compared with actin as an internal loading control. Lane a: untreated; lanes b to e: 1, 5, 10, and 20 μ mol/L HHPX, respectively. The fold changes in HO1 (relative to actin) are means of three independent experiments. ** P
    Figure Legend Snippet: Heme–HPX induces HO1 expression in primary cortical neurons. Embryonic cortical neurons were incubated for 6 h with HHPX. HO1 induction was measured by RT-PCR ( A ) to analyze the relative level of HO1 mRNA or by Western blot ( B ) to determine HO1 protein, both compared with actin as an internal loading control. Lane a: untreated; lanes b to e: 1, 5, 10, and 20 μ mol/L HHPX, respectively. The fold changes in HO1 (relative to actin) are means of three independent experiments. ** P

    Techniques Used: Expressing, Incubation, Reverse Transcription Polymerase Chain Reaction, Western Blot

    2) Product Images from "Active induction of experimental autoimmune encephalomyelitis by MOG35-55 peptide immunization is associated with differential responses in separate compartments of the choroid plexus"

    Article Title: Active induction of experimental autoimmune encephalomyelitis by MOG35-55 peptide immunization is associated with differential responses in separate compartments of the choroid plexus

    Journal: Fluids and Barriers of the CNS

    doi: 10.1186/2045-8118-9-15

    Immuno-LCM allows retrieval of tissue from specific CP compartments. A) Evidence of histological purity. Immunofluorescence was performed using FITC-conjugated pan-cytokeratin antibody to highlight the CP epithelium (green), while immunohistochemistry using alkaline phosphatase detection with NBT/BCIP as substrate was carried-out to label the endothelium of CP stromal capillaries (dark brown). LCM was performed on a Pixcell IIe LCM unit. Images both BEFORE and AFTER LCM, as well as LCM retrieved tissue deposited on the cap, are shown to highlight selective retrieval of CP stromal capillary (top row) and CP choroidal epithelial tissues (bottom row). B) Evidence of purity by qrt-PCR. Levels of CD31, an endothelial marker, and Cytokeratin-8, an epithelial marker, were probed to determine the purity of the CP capillary and CP epithelial tissues, respectively, retrieved by LCM.
    Figure Legend Snippet: Immuno-LCM allows retrieval of tissue from specific CP compartments. A) Evidence of histological purity. Immunofluorescence was performed using FITC-conjugated pan-cytokeratin antibody to highlight the CP epithelium (green), while immunohistochemistry using alkaline phosphatase detection with NBT/BCIP as substrate was carried-out to label the endothelium of CP stromal capillaries (dark brown). LCM was performed on a Pixcell IIe LCM unit. Images both BEFORE and AFTER LCM, as well as LCM retrieved tissue deposited on the cap, are shown to highlight selective retrieval of CP stromal capillary (top row) and CP choroidal epithelial tissues (bottom row). B) Evidence of purity by qrt-PCR. Levels of CD31, an endothelial marker, and Cytokeratin-8, an epithelial marker, were probed to determine the purity of the CP capillary and CP epithelial tissues, respectively, retrieved by LCM.

    Techniques Used: Laser Capture Microdissection, Immunofluorescence, Immunohistochemistry, Quantitative RT-PCR, Marker

    3) Product Images from "Nuclear factor kappa B modulates apoptosis in the brain endothelial cells and intravascular leukocytes of fatal cerebral malaria"

    Article Title: Nuclear factor kappa B modulates apoptosis in the brain endothelial cells and intravascular leukocytes of fatal cerebral malaria

    Journal: Malaria Journal

    doi: 10.1186/1475-2875-12-260

    Pattern of immunohistochemical staining for cleaved caspase-3 in the brains of fatal CM cases. A strong neuronal immunoreactivity for cleaved caspase-3 was observed in the neurons (arrows) (A) and glial cells (arrowheads) (B and C) compared with the neurons (D) and glial cells (E) of the normal brain. Scale bars = 20 μm.
    Figure Legend Snippet: Pattern of immunohistochemical staining for cleaved caspase-3 in the brains of fatal CM cases. A strong neuronal immunoreactivity for cleaved caspase-3 was observed in the neurons (arrows) (A) and glial cells (arrowheads) (B and C) compared with the neurons (D) and glial cells (E) of the normal brain. Scale bars = 20 μm.

    Techniques Used: Immunohistochemistry, Staining

    Immunohistochemical staining for cleaved caspase-3 in the blood vessel of fatal CM brain. A micrograph to demonstrate strong positive staining for cleaved caspase-3 in the ECs (arrows) sequestered with PRBCs and leukocytes (arrowheads) inside the vascular lumen. VL = vascular lumen. Scale bars = 20 μm.
    Figure Legend Snippet: Immunohistochemical staining for cleaved caspase-3 in the blood vessel of fatal CM brain. A micrograph to demonstrate strong positive staining for cleaved caspase-3 in the ECs (arrows) sequestered with PRBCs and leukocytes (arrowheads) inside the vascular lumen. VL = vascular lumen. Scale bars = 20 μm.

    Techniques Used: Immunohistochemistry, Staining

    Quantification of cleaved caspase-3 expression in neurons (A) and glial cells (B) in the brain of the control, NCM, and CM groups. a Significance of p
    Figure Legend Snippet: Quantification of cleaved caspase-3 expression in neurons (A) and glial cells (B) in the brain of the control, NCM, and CM groups. a Significance of p

    Techniques Used: Expressing

    Quantification of cleaved caspase-3 in vascular ECs and intravascular leukocytes in the brain of control, NCM and CM groups. Data were analysed as apoptotic index in the vascular ECs (A) and intravascular leukocytes (B) . a Significance of p
    Figure Legend Snippet: Quantification of cleaved caspase-3 in vascular ECs and intravascular leukocytes in the brain of control, NCM and CM groups. Data were analysed as apoptotic index in the vascular ECs (A) and intravascular leukocytes (B) . a Significance of p

    Techniques Used:

    Immunohistochemical staining for cleaved caspase-3 in the area of haemorrhage in the brains of fatal CM cases. (A) Representative photograph of area of congestion and ring haemorrhage is shown with H E stain. (B) Many glial cells located around and within the area of haemorrhage show strong positive staining for cleaved caspase-3 (arrows). Scale bars = 50 μm.
    Figure Legend Snippet: Immunohistochemical staining for cleaved caspase-3 in the area of haemorrhage in the brains of fatal CM cases. (A) Representative photograph of area of congestion and ring haemorrhage is shown with H E stain. (B) Many glial cells located around and within the area of haemorrhage show strong positive staining for cleaved caspase-3 (arrows). Scale bars = 50 μm.

    Techniques Used: Immunohistochemistry, Staining

    4) Product Images from "Broad cross-reactive IgG responses elicited by adjuvanted vaccination with recombinant influenza hemagglutinin (rHA) in ferrets and mice"

    Article Title: Broad cross-reactive IgG responses elicited by adjuvanted vaccination with recombinant influenza hemagglutinin (rHA) in ferrets and mice

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0193680

    Broader cross-reactive antibody responses are induced by prime-boost-boost vaccination with Addavax adjuvanted rHA as compared to infection with A/Hong Kong/1/1968 (A/HK68 H3N2) influenza virus. ( A ). The rHA specific IgG concentrations of heterosubtypic and stalk-specific antibodies reaction induced by vaccination with adjuvant alone control, rHA combined with Addavax adjuvant (Vax I, II and III), rHA without adjuvant (Vax Ø), or infection (Inf) with A/HongKong/1/68 (H3N2) virus, were determined using a 29-panel mPlex-Flu assay, and represented as a heatmap. ( B ). The selected antibody responses against influenza virus HA of A/HK68 were plotted over time. The antibody concentrations (ng/mL) of individual HA were calculated from the standard curves generated in same assay. Serum of individual mice were harvested before priming, as well as before and post-boost 21 day. The specific antibody concentration of IgG is shown as the mean ± standard deviations (STD) of the mean of individual mice (n = 6). The two-way analysis of variance (ANOVA) statistical analysis was conducted including experimental group and time as factors. ** p
    Figure Legend Snippet: Broader cross-reactive antibody responses are induced by prime-boost-boost vaccination with Addavax adjuvanted rHA as compared to infection with A/Hong Kong/1/1968 (A/HK68 H3N2) influenza virus. ( A ). The rHA specific IgG concentrations of heterosubtypic and stalk-specific antibodies reaction induced by vaccination with adjuvant alone control, rHA combined with Addavax adjuvant (Vax I, II and III), rHA without adjuvant (Vax Ø), or infection (Inf) with A/HongKong/1/68 (H3N2) virus, were determined using a 29-panel mPlex-Flu assay, and represented as a heatmap. ( B ). The selected antibody responses against influenza virus HA of A/HK68 were plotted over time. The antibody concentrations (ng/mL) of individual HA were calculated from the standard curves generated in same assay. Serum of individual mice were harvested before priming, as well as before and post-boost 21 day. The specific antibody concentration of IgG is shown as the mean ± standard deviations (STD) of the mean of individual mice (n = 6). The two-way analysis of variance (ANOVA) statistical analysis was conducted including experimental group and time as factors. ** p

    Techniques Used: Infection, Generated, Mouse Assay, Concentration Assay

    5) Product Images from "RGC-32 regulates reactive astrocytosis and extracellular matrix deposition in experimental autoimmune encephalomyelitis"

    Article Title: RGC-32 regulates reactive astrocytosis and extracellular matrix deposition in experimental autoimmune encephalomyelitis

    Journal: Immunologic research

    doi: 10.1007/s12026-018-9011-x

    RGC-32 deficiency attenuates the clinical course of EAE and CNS infiltration. WT and RGC-32 −/− mice were immunized with MOG 35–55 and then scored for EAE. Means ± SEM of clinical EAE scores representative of three independent experiments (5 mice for each group/experiment) are shown in ( a ). Cervical spinal cords were harvested at the peak of disease from WT ( b ) and KO ( c ) mice and stained with hematoxylin and eosin. KO mice showed fewer inflammatory infiltrates than did WT mice. Original magnification: × 200. d The score for inflammation in RGC-32 −/− was decreased to 0.8 ± 0.1 when compared with WT mice (2.4 ± 0.18) (**** p
    Figure Legend Snippet: RGC-32 deficiency attenuates the clinical course of EAE and CNS infiltration. WT and RGC-32 −/− mice were immunized with MOG 35–55 and then scored for EAE. Means ± SEM of clinical EAE scores representative of three independent experiments (5 mice for each group/experiment) are shown in ( a ). Cervical spinal cords were harvested at the peak of disease from WT ( b ) and KO ( c ) mice and stained with hematoxylin and eosin. KO mice showed fewer inflammatory infiltrates than did WT mice. Original magnification: × 200. d The score for inflammation in RGC-32 −/− was decreased to 0.8 ± 0.1 when compared with WT mice (2.4 ± 0.18) (**** p

    Techniques Used: Mouse Assay, Staining

    6) Product Images from "Brain Microbial Populations in HIV/AIDS: ?-Proteobacteria Predominate Independent of Host Immune Status"

    Article Title: Brain Microbial Populations in HIV/AIDS: ?-Proteobacteria Predominate Independent of Host Immune Status

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0054673

    Bacterial detection in human brain. Autopsy-derived ODC (A), and HIV (C) brain specimens were immunolabeled with anti-peptidoglycan antibody. Peptidoglycan (PGN)-positive bodies (arrows) were morphologically consistent with bacteria and smaller than CD45 immunopositive microglia from ODC (B) and HIV (D) patients, imaged at the same magnification. Double DIG-labeled EUB 338 probe in situ hybridization (ISH) against the 16 s rRNA gene was hybridized with slides from the same ODC (E) and HIV (G) patients and labeled with alkaline phosphatase-conjugated sheep anti-DIG FAB` fragments and stained with NBT/BCIP. ISH-positive bodies featured morphology resembling bacteria (arrow heads) and were smaller than GFAP immunopositive astrocytes in ODC (F) and HIV (H) patient sections. Peptidoglycan-labeled cells with both spherical and rod morphology were observed within the brain parenchyma and in a blood vessel (I) (White arrow) of an ODC patient. Peptidoglycan immunopositive bodies were observed within the cytoplasm of GFAP-immunolabeled astrocytes (I, inset) and Iba-1 immunolabeled microglia (J, inset). Spherical clusters of EUB 338 hybridized cells J) were evident (black arrowheads). Slides from the same ODC11 (K) and HIV11 (L) patients were processed under identical conditions except that the primary antibody was omitted. A scrambled DIG-labeled probe was hybridized to slides from the ODC11 (M) and HIV11 (N) under identical conditions used for the EUB 338 probe. In all cases specific signals were not detected. (Original magnification 200×). (O) A section from the forebrain of one of the FIV-infected cats was immunostained with the anti-PGN antibody and developed with DAB with no detectable signal. (Bar: A–H, 50 microns; I, 25 microns; J, 20 microns; K–N, 50 microns) (Magnification: A–H, 200×; I, 400×; J, 600×; I and J insets, 600×).
    Figure Legend Snippet: Bacterial detection in human brain. Autopsy-derived ODC (A), and HIV (C) brain specimens were immunolabeled with anti-peptidoglycan antibody. Peptidoglycan (PGN)-positive bodies (arrows) were morphologically consistent with bacteria and smaller than CD45 immunopositive microglia from ODC (B) and HIV (D) patients, imaged at the same magnification. Double DIG-labeled EUB 338 probe in situ hybridization (ISH) against the 16 s rRNA gene was hybridized with slides from the same ODC (E) and HIV (G) patients and labeled with alkaline phosphatase-conjugated sheep anti-DIG FAB` fragments and stained with NBT/BCIP. ISH-positive bodies featured morphology resembling bacteria (arrow heads) and were smaller than GFAP immunopositive astrocytes in ODC (F) and HIV (H) patient sections. Peptidoglycan-labeled cells with both spherical and rod morphology were observed within the brain parenchyma and in a blood vessel (I) (White arrow) of an ODC patient. Peptidoglycan immunopositive bodies were observed within the cytoplasm of GFAP-immunolabeled astrocytes (I, inset) and Iba-1 immunolabeled microglia (J, inset). Spherical clusters of EUB 338 hybridized cells J) were evident (black arrowheads). Slides from the same ODC11 (K) and HIV11 (L) patients were processed under identical conditions except that the primary antibody was omitted. A scrambled DIG-labeled probe was hybridized to slides from the ODC11 (M) and HIV11 (N) under identical conditions used for the EUB 338 probe. In all cases specific signals were not detected. (Original magnification 200×). (O) A section from the forebrain of one of the FIV-infected cats was immunostained with the anti-PGN antibody and developed with DAB with no detectable signal. (Bar: A–H, 50 microns; I, 25 microns; J, 20 microns; K–N, 50 microns) (Magnification: A–H, 200×; I, 400×; J, 600×; I and J insets, 600×).

    Techniques Used: Derivative Assay, Immunolabeling, Labeling, In Situ Hybridization, Staining, Infection

    7) Product Images from "Butyrophilin-like 1 encodes an enterocyte protein that selectively regulates functional interactions with T lymphocytes"

    Article Title: Butyrophilin-like 1 encodes an enterocyte protein that selectively regulates functional interactions with T lymphocytes

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

    doi: 10.1073/pnas.1010647108

    Quantitative RT-PCR analysis of Btnl1 , -4 , and -6 mRNA ( A ) in normal mouse tissues; ( B ) in CD45 (−) small intestinal epithelial cells (IECs) versus CD45 (+) small intestinal IEL; ( C ) in large intestines harvested from three mice; and ( D ) in the
    Figure Legend Snippet: Quantitative RT-PCR analysis of Btnl1 , -4 , and -6 mRNA ( A ) in normal mouse tissues; ( B ) in CD45 (−) small intestinal epithelial cells (IECs) versus CD45 (+) small intestinal IEL; ( C ) in large intestines harvested from three mice; and ( D ) in the

    Techniques Used: Quantitative RT-PCR, Mouse Assay

    8) Product Images from "Small molecules facilitate rapid and synchronous iPSC generation"

    Article Title: Small molecules facilitate rapid and synchronous iPSC generation

    Journal: Nature methods

    doi: 10.1038/nmeth.3142

    AGi enhances and accelerates iPSC formation across different cell types. (a) Left panel: effect of AGi on reprogramming potential of different cell types. Doxycycline was withdrawn at the indicated time points (ind., induction) and colonies were assessed for alkaline phosphatase (AP) staining 3 days after doxycycline withdrawal. Right panel: Time course analysis for reprogramming potential of GMPs. Transgene-independent iPSC colonies were obtained from GMPs after 2 days of doxycycline induction in the presence of AGi, followed by 6 days of doxycycline-independent growth. (b) Day 2 iPSCs express OCT4-GFP but no longer express OKSM (mCherry negative; scale bar is 100 um) following doxycycline withdrawal. (c) Cluster analysis based upon global gene expression analysis of the indicated samples. Note that expression data from the ESC2 line was previously published 43 . (d) Chimeric mouse showing donor-derived agouti coat color contribution from iPSCs generated in (b).
    Figure Legend Snippet: AGi enhances and accelerates iPSC formation across different cell types. (a) Left panel: effect of AGi on reprogramming potential of different cell types. Doxycycline was withdrawn at the indicated time points (ind., induction) and colonies were assessed for alkaline phosphatase (AP) staining 3 days after doxycycline withdrawal. Right panel: Time course analysis for reprogramming potential of GMPs. Transgene-independent iPSC colonies were obtained from GMPs after 2 days of doxycycline induction in the presence of AGi, followed by 6 days of doxycycline-independent growth. (b) Day 2 iPSCs express OCT4-GFP but no longer express OKSM (mCherry negative; scale bar is 100 um) following doxycycline withdrawal. (c) Cluster analysis based upon global gene expression analysis of the indicated samples. Note that expression data from the ESC2 line was previously published 43 . (d) Chimeric mouse showing donor-derived agouti coat color contribution from iPSCs generated in (b).

    Techniques Used: Staining, Expressing, Derivative Assay, Generated

    Ascorbic acid and GSK3-beta inhibitor (“AGi”) act synergistically on reprogramming. (a) Schematic of inducible, secondary reprogramming system. (b) Top panel: OKSM-expressing GMPs (as indicated by mCherry fluorescence) that remain OCT4-GFP negative after 8 days of doxycycline treatment. Bottom panel: a nascent iPSC colony after 8 days of treatment with doxycycline+AGi, showing OCT4-GFP and mCherry fluorescence (scale bar is 50 μm). (c) Alkaline phosphatase staining of doxycyline-independent, MEF-derived iPSC colonies, documenting individual and synergistic effects of ascorbic acid (AA) and GSK3-beta inhibitor (GSKi) on iPSC formation. Cells were subjected to reprogramming for 9 days, at which point doxycycline and supplements were withdrawn for an additional 3 days. (d) Representative staining of NANOG-positive iPSC colonies generated with AGi (scale bar is 200 μm). A quantitative representation of reprogramming efficiency based on transgene-independent NANOG-positive clones for the indicated conditions (n=3 biological replicates, error bars represent standard deviation for three independent experiments). (e) Plating efficiency for clonal reprogramming analyses using MEFs. Values represent the mean for three independent time points and error bars represent standard deviation. (f) Clonal analysis of reprogramming efficiency for single MEFs expressing OKSM under the indicated conditions. OKSM, Oct4 , Klf4 , Sox2 , c-Myc ; mC, mCherry; MEF, murine embryonic fibroblast; GMP, granulocyte/macrophage progenitor; HSC, hematopoietic stem cell.
    Figure Legend Snippet: Ascorbic acid and GSK3-beta inhibitor (“AGi”) act synergistically on reprogramming. (a) Schematic of inducible, secondary reprogramming system. (b) Top panel: OKSM-expressing GMPs (as indicated by mCherry fluorescence) that remain OCT4-GFP negative after 8 days of doxycycline treatment. Bottom panel: a nascent iPSC colony after 8 days of treatment with doxycycline+AGi, showing OCT4-GFP and mCherry fluorescence (scale bar is 50 μm). (c) Alkaline phosphatase staining of doxycyline-independent, MEF-derived iPSC colonies, documenting individual and synergistic effects of ascorbic acid (AA) and GSK3-beta inhibitor (GSKi) on iPSC formation. Cells were subjected to reprogramming for 9 days, at which point doxycycline and supplements were withdrawn for an additional 3 days. (d) Representative staining of NANOG-positive iPSC colonies generated with AGi (scale bar is 200 μm). A quantitative representation of reprogramming efficiency based on transgene-independent NANOG-positive clones for the indicated conditions (n=3 biological replicates, error bars represent standard deviation for three independent experiments). (e) Plating efficiency for clonal reprogramming analyses using MEFs. Values represent the mean for three independent time points and error bars represent standard deviation. (f) Clonal analysis of reprogramming efficiency for single MEFs expressing OKSM under the indicated conditions. OKSM, Oct4 , Klf4 , Sox2 , c-Myc ; mC, mCherry; MEF, murine embryonic fibroblast; GMP, granulocyte/macrophage progenitor; HSC, hematopoietic stem cell.

    Techniques Used: Activated Clotting Time Assay, Expressing, Fluorescence, Staining, Derivative Assay, Generated, Clone Assay, Standard Deviation

    9) Product Images from "Nasal delivery of H5N1 avian influenza vaccine formulated with GenJet™ or in vivo-jetPEI® induces enhanced serological, cellular and protective immune responses"

    Article Title: Nasal delivery of H5N1 avian influenza vaccine formulated with GenJet™ or in vivo-jetPEI® induces enhanced serological, cellular and protective immune responses

    Journal: Drug Delivery

    doi: 10.1080/10717544.2018.1450909

    GenJet™ and in vivo -jetPEI ® enhanced the H5N1-specific T-cell responses. Balb/c mice (3–5 mice/group) were intranasally administered with A/IN/05 vaccine with or without GenJet™ or in vivo -jetPEI ® using prime-boost regimen as described in Figure 1 . (A) One week after booster immunization, the lung tissues were harvested and single cell suspensions were prepared. About 10 6 cells from the lung were stimulated in vitro with HA peptide for 6 h to examine the antigen-specific CD8 T-cell response; or with RG A/IN/05 virus at an MOI of 1 for 16 h to examine the antigen-specific CD4 T-cell response. GolgiPlug™ was added during the last 5 h of incubation. Cells were surface stained with anti-CD44, anti-CD4 or anti-CD8 antibody (BD Bioscience), followed by intracellular staining with anti-IFNγ antibody (BD Bioscience). The frequency of IFN-γ producing T cells in total activated T cells was presented. (B) One-week post-booster immunization, the draining lymph nodes, lungs and spleen tissues were harvested and the frequency of HA518-specific CD8 T cells in total activated CD8 T cells was stained using H-2K d /IYSTVASSL tetramer. (C) Three weeks after booster immunization, sera were collected and IgG2a, IgG2b IgG3 and IgG1 antibodies against A/IN/05 were assessed by ELISA. The data are representative of two independent experiments (3–5 mice each group) and error bars represent SEM. One-way ANOVA with Bonferroni post-analysis was used to analyze differences among treatments. p
    Figure Legend Snippet: GenJet™ and in vivo -jetPEI ® enhanced the H5N1-specific T-cell responses. Balb/c mice (3–5 mice/group) were intranasally administered with A/IN/05 vaccine with or without GenJet™ or in vivo -jetPEI ® using prime-boost regimen as described in Figure 1 . (A) One week after booster immunization, the lung tissues were harvested and single cell suspensions were prepared. About 10 6 cells from the lung were stimulated in vitro with HA peptide for 6 h to examine the antigen-specific CD8 T-cell response; or with RG A/IN/05 virus at an MOI of 1 for 16 h to examine the antigen-specific CD4 T-cell response. GolgiPlug™ was added during the last 5 h of incubation. Cells were surface stained with anti-CD44, anti-CD4 or anti-CD8 antibody (BD Bioscience), followed by intracellular staining with anti-IFNγ antibody (BD Bioscience). The frequency of IFN-γ producing T cells in total activated T cells was presented. (B) One-week post-booster immunization, the draining lymph nodes, lungs and spleen tissues were harvested and the frequency of HA518-specific CD8 T cells in total activated CD8 T cells was stained using H-2K d /IYSTVASSL tetramer. (C) Three weeks after booster immunization, sera were collected and IgG2a, IgG2b IgG3 and IgG1 antibodies against A/IN/05 were assessed by ELISA. The data are representative of two independent experiments (3–5 mice each group) and error bars represent SEM. One-way ANOVA with Bonferroni post-analysis was used to analyze differences among treatments. p

    Techniques Used: In Vivo, Mouse Assay, In Vitro, Incubation, Staining, Enzyme-linked Immunosorbent Assay

    GenJet™ and in vivo -jetPEI enhances the H5N1 vaccine-induced systemic antibody responses and memory B-cell responses. Balb/c mice (5 mice/group) were intranasally administered with 3 µg of A/IN/05 vaccine with or without GenJet™ (10 µl/each mouse as described in previous study (Kulkarni et al., 2014 )) or in vivo -jetPEI ® (0.6 μl/mouse according to manufacturer’s protocol). One month later, mice were boosted with the same vaccine formulations. The control mouse group received GenJet™, in vivo -jetPEI ® or PBS at both time points. (A) Three weeks after booster immunization, sera were collected and IgG, IgA and IgM antibodies against A/IN/05 were assessed by ELISA. (B) One week after booster immunization, the spleen were harvested and the frequency of A/IN/05-specific IgG + ASCs in the spleen were measured by ELISPOT assay. The number of A/IN/05-specific IgG + ASCs were normalized against the number of total IgG + secreting ASCs and presented as % Ag-specific IgG + B cells. The data are representative of two independent experiments (3–5 mice each group) and error bars represent SEM. One-way ANOVA with Bonferroni post-analysis was used to analyze differences among different groups. p
    Figure Legend Snippet: GenJet™ and in vivo -jetPEI enhances the H5N1 vaccine-induced systemic antibody responses and memory B-cell responses. Balb/c mice (5 mice/group) were intranasally administered with 3 µg of A/IN/05 vaccine with or without GenJet™ (10 µl/each mouse as described in previous study (Kulkarni et al., 2014 )) or in vivo -jetPEI ® (0.6 μl/mouse according to manufacturer’s protocol). One month later, mice were boosted with the same vaccine formulations. The control mouse group received GenJet™, in vivo -jetPEI ® or PBS at both time points. (A) Three weeks after booster immunization, sera were collected and IgG, IgA and IgM antibodies against A/IN/05 were assessed by ELISA. (B) One week after booster immunization, the spleen were harvested and the frequency of A/IN/05-specific IgG + ASCs in the spleen were measured by ELISPOT assay. The number of A/IN/05-specific IgG + ASCs were normalized against the number of total IgG + secreting ASCs and presented as % Ag-specific IgG + B cells. The data are representative of two independent experiments (3–5 mice each group) and error bars represent SEM. One-way ANOVA with Bonferroni post-analysis was used to analyze differences among different groups. p

    Techniques Used: In Vivo, Mouse Assay, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot

    10) Product Images from "Ash2l interacts with Oct4-stemness circuitry to promote super-enhancer-driven pluripotency network"

    Article Title: Ash2l interacts with Oct4-stemness circuitry to promote super-enhancer-driven pluripotency network

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkz801

    Ash2l binding motif is crucial for the recruitment of OSN to Jarid2, Oct4 and Nanog super-enhancers. ( A ) Schematic illustration of the design of sequence-specific sgRNA for blocking of Jarid2, Nanog or Oct4 super-enhancers using monoallelic silencing (labeled as sgRNA AB ) or biallelic silencing (labeled as sgRNA AC , sgRNA DE , sgRNA DF , sgRNA GH and sgRNA GI ). The dCas9 nuclease is targeted to super-enhancer of Jarid2, Nanog or Oct4 by either set of sgRNA. Three amplicons for each enhancer (Jarid2: R1: enhancer; R2: promoter, R3: desert; Nanog: R4: enhancer; R5: promoter, R6: desert; Oct4: R7: enhancer; R8: promoter, R9: desert) were designed to evaluate protein enrichment in ChIP-qPCR assay. ( B ) ChIP-qPCR analysis showing the enrichment of indicated protein at Jarid2, Nanog and Oct4 super-enhancers in ESCs with CRISPRi/dCas9-mediated interference of Ash2l binding. ChIP-qPCR analysis with indicated antibodies was conducted in CRISPRi/dCas9-modified ESCs, and the enrichment of each protein at R1–3 regions, R4–6 regions or R7-R9 regions was evaluated with specific primers. Data showed that CRISPRi/dCas9-mediated interference of Ash2l binding at Jarid2 enhancer (upper), Nanog enhancer (middle) and Oct4 enhancer (lower), specifically increased dCas9 binding and decreased the binding of Ash2l, Oct4, Sox2, Nanog and H3K27ac, to R1, R4 and R7, but not R2, R3, R5, R6, R8 and R9. ( C ) qPCR results showing the effect of CRISPRi/dCas9-mediated interference of Ash2l binding on mRNA expression of Jarid2, Nanog and Oct4. ( D ) Western blot showing the effect of CRISPRi/dCas9-mediated interference of Ash2l binding on the protein content of Jarid2, Nanog and Oct4. ( E ) qPCR shows CRISPRi/dCas9-mediated interference of Ash2l binding at indicated enhancers abrogate the upregulation of Jarid2, Nanog, Oct4 transcripts induced by Ash2l overexpression. ( F ) Reporter assay shows that Flag-Ash2l and Myc-Oct4 induce a synergistic effect on enhancer activity in Jarid2, Nanog and Oct4 super-enhancers. The Ash2l-mediated enhancement of Jarid2, Nanog and Oct4 enhancer activation can be abrogated by specific sgRNA for indicated enhancer. Data are presented as mean ± SD; *, P
    Figure Legend Snippet: Ash2l binding motif is crucial for the recruitment of OSN to Jarid2, Oct4 and Nanog super-enhancers. ( A ) Schematic illustration of the design of sequence-specific sgRNA for blocking of Jarid2, Nanog or Oct4 super-enhancers using monoallelic silencing (labeled as sgRNA AB ) or biallelic silencing (labeled as sgRNA AC , sgRNA DE , sgRNA DF , sgRNA GH and sgRNA GI ). The dCas9 nuclease is targeted to super-enhancer of Jarid2, Nanog or Oct4 by either set of sgRNA. Three amplicons for each enhancer (Jarid2: R1: enhancer; R2: promoter, R3: desert; Nanog: R4: enhancer; R5: promoter, R6: desert; Oct4: R7: enhancer; R8: promoter, R9: desert) were designed to evaluate protein enrichment in ChIP-qPCR assay. ( B ) ChIP-qPCR analysis showing the enrichment of indicated protein at Jarid2, Nanog and Oct4 super-enhancers in ESCs with CRISPRi/dCas9-mediated interference of Ash2l binding. ChIP-qPCR analysis with indicated antibodies was conducted in CRISPRi/dCas9-modified ESCs, and the enrichment of each protein at R1–3 regions, R4–6 regions or R7-R9 regions was evaluated with specific primers. Data showed that CRISPRi/dCas9-mediated interference of Ash2l binding at Jarid2 enhancer (upper), Nanog enhancer (middle) and Oct4 enhancer (lower), specifically increased dCas9 binding and decreased the binding of Ash2l, Oct4, Sox2, Nanog and H3K27ac, to R1, R4 and R7, but not R2, R3, R5, R6, R8 and R9. ( C ) qPCR results showing the effect of CRISPRi/dCas9-mediated interference of Ash2l binding on mRNA expression of Jarid2, Nanog and Oct4. ( D ) Western blot showing the effect of CRISPRi/dCas9-mediated interference of Ash2l binding on the protein content of Jarid2, Nanog and Oct4. ( E ) qPCR shows CRISPRi/dCas9-mediated interference of Ash2l binding at indicated enhancers abrogate the upregulation of Jarid2, Nanog, Oct4 transcripts induced by Ash2l overexpression. ( F ) Reporter assay shows that Flag-Ash2l and Myc-Oct4 induce a synergistic effect on enhancer activity in Jarid2, Nanog and Oct4 super-enhancers. The Ash2l-mediated enhancement of Jarid2, Nanog and Oct4 enhancer activation can be abrogated by specific sgRNA for indicated enhancer. Data are presented as mean ± SD; *, P

    Techniques Used: Binding Assay, Sequencing, Blocking Assay, Labeling, Protein Enrichment, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Modification, Expressing, Western Blot, Over Expression, Reporter Assay, Activity Assay, Activation Assay

    11) Product Images from "High-Resolution Temporal Response Patterns to Influenza Vaccine Reveal a Distinct Human Plasma Cell Gene Signature"

    Article Title: High-Resolution Temporal Response Patterns to Influenza Vaccine Reveal a Distinct Human Plasma Cell Gene Signature

    Journal: Scientific Reports

    doi: 10.1038/srep02327

    Subject-specific B cell vaccine response characteristics. (a) A single gene expression eigenfunction accounted for 90% of sample variation in each subject vaccinated in the previous three years (S02, S04, and S04). Multiple eigenfunctions were found in RNA Seq data of subjects not previously vaccinated (S05, S06) (full list of significant genes in Supplementary Table S3 ). (b) By HAI titer, S02, S03 and S04 had previous immunity to two HA antigens. S02 had increases in antibody levels at least 24 hours before S03 and S04 (p-values, Supplementary Table S2 ). (c) Anti-vaccine IgM, IgG, and IgA levels parallel changes in HAI titer (p-values Supplementary Table S2 ). (d) ELISPOT assays show functional vaccine-specific IgA, IgG, and IgM ASC increase as serum antibody levels increase. (e) Phenotypic changes of CD3 − CD19 + CD20 + CD27 − naïve B cells, CD19 + CD27 + CD20 + CD38 + CD138 − memory B cells, CD19 + CD27 ++ CD38 ++ CD138 − plasmablasts and CD19 lo CD20 lo CD27 ++ CD38 ++ CD138 + plasma cells. Plasmablast and plasma cell populations increase as ASC appear (p-values Supplementary Table S4 ). (f) Expression of genes (ratio to day 0) change as expected based on available literature in S02, S03 and, S04 (p-values, Table S3 ). CD5 and SERPINB9 levels decrease at peak response consistent with a fractional decrease in naïve and cytolytic B cells. Fewer genes changed significantly in S06 and S05, subjects reporting no vaccination in the previous 3 years. S02 showed peak response at day 5, earlier than S03 and S04. (N/D = no data).
    Figure Legend Snippet: Subject-specific B cell vaccine response characteristics. (a) A single gene expression eigenfunction accounted for 90% of sample variation in each subject vaccinated in the previous three years (S02, S04, and S04). Multiple eigenfunctions were found in RNA Seq data of subjects not previously vaccinated (S05, S06) (full list of significant genes in Supplementary Table S3 ). (b) By HAI titer, S02, S03 and S04 had previous immunity to two HA antigens. S02 had increases in antibody levels at least 24 hours before S03 and S04 (p-values, Supplementary Table S2 ). (c) Anti-vaccine IgM, IgG, and IgA levels parallel changes in HAI titer (p-values Supplementary Table S2 ). (d) ELISPOT assays show functional vaccine-specific IgA, IgG, and IgM ASC increase as serum antibody levels increase. (e) Phenotypic changes of CD3 − CD19 + CD20 + CD27 − naïve B cells, CD19 + CD27 + CD20 + CD38 + CD138 − memory B cells, CD19 + CD27 ++ CD38 ++ CD138 − plasmablasts and CD19 lo CD20 lo CD27 ++ CD38 ++ CD138 + plasma cells. Plasmablast and plasma cell populations increase as ASC appear (p-values Supplementary Table S4 ). (f) Expression of genes (ratio to day 0) change as expected based on available literature in S02, S03 and, S04 (p-values, Table S3 ). CD5 and SERPINB9 levels decrease at peak response consistent with a fractional decrease in naïve and cytolytic B cells. Fewer genes changed significantly in S06 and S05, subjects reporting no vaccination in the previous 3 years. S02 showed peak response at day 5, earlier than S03 and S04. (N/D = no data).

    Techniques Used: Expressing, RNA Sequencing Assay, Enzyme-linked Immunospot, Functional Assay

    12) Product Images from "Colorimetric Focus-Forming Assay with Automated Focus Counting by Image Analysis for Quantification of Infectious Hepatitis C Virions"

    Article Title: Colorimetric Focus-Forming Assay with Automated Focus Counting by Image Analysis for Quantification of Infectious Hepatitis C Virions

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0043960

    Comparison of colorimetric focus-forming assays using various secondary antibodies and chromogenic substrates. (A) Huh-7.5 cells inoculated with serial dilutions of HCV were immunostained with monoclonal anti-HCV core antibody and secondary antibodies conjugated with different enzymes, as indicated, followed by chromogenic development using various substrates, and image scanning by ELISpot reader. (B) A magnified view of the scanned image of the colorimetric focus-forming assay revealed that alkaline phosphatase-conjugated secondary antibody with BCIP/NBT yielded the best results, considering background and distinctness of the foci. Biotin-2°Ab, biotin-conjugated secondary antibody; SA-AP, streptavidin-conjugated alkaline phosphatase; AP-2°Ab, alkaline phosphatase-conjugated secondary antibody; HRP-2°Ab, horseradish peroxidase-conjugated secondary antibody; BCIP/NBT, 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium; DAB, 3,3′-diaminobenzidine; TMB, 3,3′,5,5′-tetramethylbenzidine.
    Figure Legend Snippet: Comparison of colorimetric focus-forming assays using various secondary antibodies and chromogenic substrates. (A) Huh-7.5 cells inoculated with serial dilutions of HCV were immunostained with monoclonal anti-HCV core antibody and secondary antibodies conjugated with different enzymes, as indicated, followed by chromogenic development using various substrates, and image scanning by ELISpot reader. (B) A magnified view of the scanned image of the colorimetric focus-forming assay revealed that alkaline phosphatase-conjugated secondary antibody with BCIP/NBT yielded the best results, considering background and distinctness of the foci. Biotin-2°Ab, biotin-conjugated secondary antibody; SA-AP, streptavidin-conjugated alkaline phosphatase; AP-2°Ab, alkaline phosphatase-conjugated secondary antibody; HRP-2°Ab, horseradish peroxidase-conjugated secondary antibody; BCIP/NBT, 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium; DAB, 3,3′-diaminobenzidine; TMB, 3,3′,5,5′-tetramethylbenzidine.

    Techniques Used: Enzyme-linked Immunospot, Focus Forming Assay

    13) Product Images from "SMRTe, a silencing mediator for retinoid and thyroid hormone receptors-extended isoform that is more related to the nuclear receptor corepressor"

    Article Title: SMRTe, a silencing mediator for retinoid and thyroid hormone receptors-extended isoform that is more related to the nuclear receptor corepressor

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

    doi:

    Detection of endogenous SMRT proteins. HeLa nuclear extract, together with in vitro -translated [ 35 S]methionine-labeled N-CoR and C-SMRT, were separated on a SDS/PAGE. The N-CoR and C-SMRT polypeptides were detected by autoradiography ( Left ). An identical gel was processed for Western blotting using an affinity-purified rabbit anti-C-SMRT polyclonal antibody and detected by 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitroblue tetrazolium (NBT) color reaction ( Center ). One major polypeptide similar to the size of N-CoR (270 kDa) was detected in the HeLa nuclear extract, in addition to two minor bands of 180 and 80 kDa, respectively (arrows). The anti-SMRT antibody does not crossreact with N-CoR. The same HeLa nuclear extract also was processed for Western blotting using anti-C-SMRT antibody but developed by ECL + reaction ( Right ). The three specific SMRT polypeptides and two nonspecific bands (open arrowheads) below 80 kDa were indicated.
    Figure Legend Snippet: Detection of endogenous SMRT proteins. HeLa nuclear extract, together with in vitro -translated [ 35 S]methionine-labeled N-CoR and C-SMRT, were separated on a SDS/PAGE. The N-CoR and C-SMRT polypeptides were detected by autoradiography ( Left ). An identical gel was processed for Western blotting using an affinity-purified rabbit anti-C-SMRT polyclonal antibody and detected by 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitroblue tetrazolium (NBT) color reaction ( Center ). One major polypeptide similar to the size of N-CoR (270 kDa) was detected in the HeLa nuclear extract, in addition to two minor bands of 180 and 80 kDa, respectively (arrows). The anti-SMRT antibody does not crossreact with N-CoR. The same HeLa nuclear extract also was processed for Western blotting using anti-C-SMRT antibody but developed by ECL + reaction ( Right ). The three specific SMRT polypeptides and two nonspecific bands (open arrowheads) below 80 kDa were indicated.

    Techniques Used: In Vitro, Labeling, SDS Page, Autoradiography, Western Blot, Affinity Purification

    14) Product Images from "Ig? allelic inclusion is a consequence of receptor editing"

    Article Title: Ig? allelic inclusion is a consequence of receptor editing

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20061918

    Allelically included B cells result from receptor editing. (A) Kinetics of bone marrow development of included and excluded B cells. Linear regression analysis shows the percentage of B220 low BrdU + B cells plotted against time. Percentage values of excluded mCκ + (blue ovals) and hCκ + (green squares) cells are represented in the left y axis, and the percentage of mCκ + hCκ + -included cells (red circles) is depicted in the right y axis. Igκ m/h mice were injected with 0.5 mg of BrdU intraperitoneally and killed after 6, 12, 18, 24, and 48 h (three mice per time point). Cells were permeabilized and stained with anti–BrdU-APC, mCκ-PE, hCκ-FITC, and B220-PerCP antibodies. (B) Comparative analysis of Jκ usage (percentage) in allelically included (red bars; n = 168 transcripts) and excluded (blue bars; n = 233 transcripts) B cells. (C) Antibodies purified from the supernatants of 15 mCκ + hCκ + hybridoma clones were compared with 38 antibodies from hybridomas expressing only one allele for binding to HEp-2 cells and double-stranded DNA. HEp-2 binding was compared with positive and negative control sera provided by the manufacturer. To ensure HEp-2 binding was not caused by xenoreactivity, self-specificities were verified by a commercial ANA assay designed for mice (not depicted).
    Figure Legend Snippet: Allelically included B cells result from receptor editing. (A) Kinetics of bone marrow development of included and excluded B cells. Linear regression analysis shows the percentage of B220 low BrdU + B cells plotted against time. Percentage values of excluded mCκ + (blue ovals) and hCκ + (green squares) cells are represented in the left y axis, and the percentage of mCκ + hCκ + -included cells (red circles) is depicted in the right y axis. Igκ m/h mice were injected with 0.5 mg of BrdU intraperitoneally and killed after 6, 12, 18, 24, and 48 h (three mice per time point). Cells were permeabilized and stained with anti–BrdU-APC, mCκ-PE, hCκ-FITC, and B220-PerCP antibodies. (B) Comparative analysis of Jκ usage (percentage) in allelically included (red bars; n = 168 transcripts) and excluded (blue bars; n = 233 transcripts) B cells. (C) Antibodies purified from the supernatants of 15 mCκ + hCκ + hybridoma clones were compared with 38 antibodies from hybridomas expressing only one allele for binding to HEp-2 cells and double-stranded DNA. HEp-2 binding was compared with positive and negative control sera provided by the manufacturer. To ensure HEp-2 binding was not caused by xenoreactivity, self-specificities were verified by a commercial ANA assay designed for mice (not depicted).

    Techniques Used: Mouse Assay, Injection, Staining, Purification, Clone Assay, Expressing, Binding Assay, Negative Control

    15) Product Images from "Microglial Activation and Antioxidant Responses Induced by the Parkinson's Disease Protein ?-Synuclein"

    Article Title: Microglial Activation and Antioxidant Responses Induced by the Parkinson's Disease Protein ?-Synuclein

    Journal: Journal of Neuroimmune Pharmacology

    doi: 10.1007/s11481-012-9401-0

    Exposure of primary microglia to α-synuclein increases antioxidant expression. Primary microglia from ARE transgenic mice were histochemically stained with BCIP/NBT ( purple ) to detect hPLAP activity and nuclear red counterstained ( pink ). Microglia exposed to Buffer ( a , b ) or Buffer DA ( e , f ) displayed less phosphatase activity than SYN-treated cells and also exhibited the prototypic morphology of resting microglia (dashed arrows; a , b , e , f i ) with a few glia that were activated but not phagocytic (solid arrows). In contrast, cells exposed to SYN or SYN DA displayed increased numbers of microglia with the characteristic amoeboid morphology of phagocytic microglia compared to the other exposure paradigms (solid arrowheads; c , d , g , h i ). Interestingly, microglia exposed to SYN DA had the greatest percentage of microglia expressing phosphatase activity with nearly 75 % of the treated microglia activated, as demonstrated by thickened processes and ameboid shape (solid arrow and solid arrowhead, respectively; g , h i ). Scale bar for 10x ( a , c , e , g ) and 40x ( b , d , f , h ) images represent 100 µm and 20 µm respectively. Boxes in a , c , e , and g denote the area for 40x images b , d , f and h . Cell counts were performed on nine random 20x images from each sample and categorized based on staining and morphology as outlined in Materials and Methods ( i )
    Figure Legend Snippet: Exposure of primary microglia to α-synuclein increases antioxidant expression. Primary microglia from ARE transgenic mice were histochemically stained with BCIP/NBT ( purple ) to detect hPLAP activity and nuclear red counterstained ( pink ). Microglia exposed to Buffer ( a , b ) or Buffer DA ( e , f ) displayed less phosphatase activity than SYN-treated cells and also exhibited the prototypic morphology of resting microglia (dashed arrows; a , b , e , f i ) with a few glia that were activated but not phagocytic (solid arrows). In contrast, cells exposed to SYN or SYN DA displayed increased numbers of microglia with the characteristic amoeboid morphology of phagocytic microglia compared to the other exposure paradigms (solid arrowheads; c , d , g , h i ). Interestingly, microglia exposed to SYN DA had the greatest percentage of microglia expressing phosphatase activity with nearly 75 % of the treated microglia activated, as demonstrated by thickened processes and ameboid shape (solid arrow and solid arrowhead, respectively; g , h i ). Scale bar for 10x ( a , c , e , g ) and 40x ( b , d , f , h ) images represent 100 µm and 20 µm respectively. Boxes in a , c , e , and g denote the area for 40x images b , d , f and h . Cell counts were performed on nine random 20x images from each sample and categorized based on staining and morphology as outlined in Materials and Methods ( i )

    Techniques Used: Expressing, Transgenic Assay, Mouse Assay, Staining, Activity Assay

    Antioxidant responses in vivo . The SNpc of 1-, 6- and 12-month old ARE and SARE mice ( n = 4/genotype/age) were processed for hPLAP activity measurements using a BCIP/NBT staining protocol. a Image of phosphatase activity in the SNpc of an SARE mouse (12-months of age) demonstrating robust activity in this region of the brain (white arrows). Images were taken at 10X (scale bar = 100 μm) magnification. b The density of phosphatase activity was determined from the SNpc of BCIP/NBT stained tissue for all mice and reported as pixels/mm 2 . There is no statistically significant difference between α-synuclein overexpressing mice (SARE) and ARE mice in phosphatase activity
    Figure Legend Snippet: Antioxidant responses in vivo . The SNpc of 1-, 6- and 12-month old ARE and SARE mice ( n = 4/genotype/age) were processed for hPLAP activity measurements using a BCIP/NBT staining protocol. a Image of phosphatase activity in the SNpc of an SARE mouse (12-months of age) demonstrating robust activity in this region of the brain (white arrows). Images were taken at 10X (scale bar = 100 μm) magnification. b The density of phosphatase activity was determined from the SNpc of BCIP/NBT stained tissue for all mice and reported as pixels/mm 2 . There is no statistically significant difference between α-synuclein overexpressing mice (SARE) and ARE mice in phosphatase activity

    Techniques Used: In Vivo, Mouse Assay, Activity Assay, Staining

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    Article Snippet: .. Alkaline phosphatase staining Alkaline phosphatase kits (Vector Labs) were used according to the manufacturer’s recommendations to assess pluripotency. .. Prior to staining, doxycycline and cell culture supplements were removed for a minimum of 3–4 days to eliminate exogenous OKSM expression.

    Article Title: Active induction of experimental autoimmune encephalomyelitis by MOG35-55 peptide immunization is associated with differential responses in separate compartments of the choroid plexus
    Article Snippet: Immunostaining for Immuno-LCM Immunostaining was performed as detailed [ , , ], with minor modifications. .. The CP stromal capillaries were stained using alkaline phosphatase substrate NBT (nitro-blue tetrazolium chloride)/BCIP (5-bromo-4-chloro-3'-indolyphosphate p-toluidine salt), (Vector Labs, Burlingame, CA) for 3–5 minutes in 100 Mm Tris–HCl (pH 9.5) to detect endogenous alkaline phosphatase activity in the endothelial cells. ..

    Article Title: Butyrophilin-like 1 encodes an enterocyte protein that selectively regulates functional interactions with T lymphocytes
    Article Snippet: Acetone-fixed frozen sections or Methanol-Carnoy's fixed and paraffin-embedded sections were stained with anti-Btnl1 only or double stained for expression of Btnl1 and CD3. .. The alkaline phosphatase conjugated anti-CD3 staining was developed with Vector Blue Alkaline Phosphatase Substrate Kit III (Vector Laboratories) and peroxidase stained Btnl1 sections developed using the 3, 3′-diaminobenzidine substrate kit (Vector Laboratories). .. Frozen sections or Methanol-Carnoy's fixed and paraffin embedded sections were double stained for Btnl1 and CD3 or Btnl1 and A33.

    Activity Assay:

    Article Title: Active induction of experimental autoimmune encephalomyelitis by MOG35-55 peptide immunization is associated with differential responses in separate compartments of the choroid plexus
    Article Snippet: Immunostaining for Immuno-LCM Immunostaining was performed as detailed [ , , ], with minor modifications. .. The CP stromal capillaries were stained using alkaline phosphatase substrate NBT (nitro-blue tetrazolium chloride)/BCIP (5-bromo-4-chloro-3'-indolyphosphate p-toluidine salt), (Vector Labs, Burlingame, CA) for 3–5 minutes in 100 Mm Tris–HCl (pH 9.5) to detect endogenous alkaline phosphatase activity in the endothelial cells. ..

    Plasmid Preparation:

    Article Title: Heme–hemopexin complex attenuates neuronal cell death and stroke damage
    Article Snippet: This antibody cross-reacts with mouse HPX and the secondary antibody was selected accordingly (Vector Laboratories). .. The HPX was finally visualized by developing the sections with the Vector Blue Alkaline Phosphatase Substrate Kit III (Vector Laboratories). .. Cultured embryonic neurons were grown on poly-D-lysine-coated glass coverslips for 10 days.

    Article Title: RGC-32 regulates reactive astrocytosis and extracellular matrix deposition in experimental autoimmune encephalomyelitis
    Article Snippet: Frozen sections (5 μm) from the brains of adult patients with MS were double-stained for collagen I, IV, or V and for GFAP as previously described [ ]. .. Cryosections were initially treated with Bloxall (Vector Labs) to remove endogenous peroxidase and alkaline phosphatase, then incubated with primary antibodies against collagen I, IV, or V as described above, and the reaction was developed with Nova RED (Vector Labs) for collagen I and RGC-32 or Vector alkaline phosphatase substrate kit III (Vector Labs) for collagens IV and V. The sections were then incubated overnight with mouse IgG anti-GFAP monoclonal antibody (eBioscience, San Diego, CA). .. The slides were washed several times in PBS and reacted with alkaline phosphatase-conjugated goat anti-mouse (Sigma-Aldrich), diluted 1/400 and processed with Vector alkaline phosphatase substrate kit III (Vector Labs) or Nova RED as the chromogen substrate.

    Article Title: Butyrophilin-like 1 encodes an enterocyte protein that selectively regulates functional interactions with T lymphocytes
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    Incubation:

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    Article Title: Nuclear factor kappa B modulates apoptosis in the brain endothelial cells and intravascular leukocytes of fatal cerebral malaria
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    Avidin-Biotin Assay:

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    Vector Laboratories vector blue alkaline phosphatase substrate kit iii
    <t>Heme–HPX</t> induces HO1 expression in primary cortical neurons. Embryonic cortical neurons were incubated for 6 h with HHPX. HO1 induction was measured by RT-PCR ( A ) to analyze the relative level of HO1 mRNA or by Western blot ( B ) to determine HO1 protein, both compared with actin as an internal loading control. Lane a: untreated; lanes b to e: 1, 5, 10, and 20 μ mol/L HHPX, respectively. The fold changes in HO1 (relative to actin) are means of <t>three</t> independent experiments. ** P
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    Heme–HPX induces HO1 expression in primary cortical neurons. Embryonic cortical neurons were incubated for 6 h with HHPX. HO1 induction was measured by RT-PCR ( A ) to analyze the relative level of HO1 mRNA or by Western blot ( B ) to determine HO1 protein, both compared with actin as an internal loading control. Lane a: untreated; lanes b to e: 1, 5, 10, and 20 μ mol/L HHPX, respectively. The fold changes in HO1 (relative to actin) are means of three independent experiments. ** P

    Journal: Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism

    Article Title: Heme–hemopexin complex attenuates neuronal cell death and stroke damage

    doi: 10.1038/jcbfm.2009.19

    Figure Lengend Snippet: Heme–HPX induces HO1 expression in primary cortical neurons. Embryonic cortical neurons were incubated for 6 h with HHPX. HO1 induction was measured by RT-PCR ( A ) to analyze the relative level of HO1 mRNA or by Western blot ( B ) to determine HO1 protein, both compared with actin as an internal loading control. Lane a: untreated; lanes b to e: 1, 5, 10, and 20 μ mol/L HHPX, respectively. The fold changes in HO1 (relative to actin) are means of three independent experiments. ** P

    Article Snippet: The HPX was finally visualized by developing the sections with the Vector Blue Alkaline Phosphatase Substrate Kit III (Vector Laboratories).

    Techniques: Expressing, Incubation, Reverse Transcription Polymerase Chain Reaction, Western Blot

    Immuno-LCM allows retrieval of tissue from specific CP compartments. A) Evidence of histological purity. Immunofluorescence was performed using FITC-conjugated pan-cytokeratin antibody to highlight the CP epithelium (green), while immunohistochemistry using alkaline phosphatase detection with NBT/BCIP as substrate was carried-out to label the endothelium of CP stromal capillaries (dark brown). LCM was performed on a Pixcell IIe LCM unit. Images both BEFORE and AFTER LCM, as well as LCM retrieved tissue deposited on the cap, are shown to highlight selective retrieval of CP stromal capillary (top row) and CP choroidal epithelial tissues (bottom row). B) Evidence of purity by qrt-PCR. Levels of CD31, an endothelial marker, and Cytokeratin-8, an epithelial marker, were probed to determine the purity of the CP capillary and CP epithelial tissues, respectively, retrieved by LCM.

    Journal: Fluids and Barriers of the CNS

    Article Title: Active induction of experimental autoimmune encephalomyelitis by MOG35-55 peptide immunization is associated with differential responses in separate compartments of the choroid plexus

    doi: 10.1186/2045-8118-9-15

    Figure Lengend Snippet: Immuno-LCM allows retrieval of tissue from specific CP compartments. A) Evidence of histological purity. Immunofluorescence was performed using FITC-conjugated pan-cytokeratin antibody to highlight the CP epithelium (green), while immunohistochemistry using alkaline phosphatase detection with NBT/BCIP as substrate was carried-out to label the endothelium of CP stromal capillaries (dark brown). LCM was performed on a Pixcell IIe LCM unit. Images both BEFORE and AFTER LCM, as well as LCM retrieved tissue deposited on the cap, are shown to highlight selective retrieval of CP stromal capillary (top row) and CP choroidal epithelial tissues (bottom row). B) Evidence of purity by qrt-PCR. Levels of CD31, an endothelial marker, and Cytokeratin-8, an epithelial marker, were probed to determine the purity of the CP capillary and CP epithelial tissues, respectively, retrieved by LCM.

    Article Snippet: The CP stromal capillaries were stained using alkaline phosphatase substrate NBT (nitro-blue tetrazolium chloride)/BCIP (5-bromo-4-chloro-3'-indolyphosphate p-toluidine salt), (Vector Labs, Burlingame, CA) for 3–5 minutes in 100 Mm Tris–HCl (pH 9.5) to detect endogenous alkaline phosphatase activity in the endothelial cells.

    Techniques: Laser Capture Microdissection, Immunofluorescence, Immunohistochemistry, Quantitative RT-PCR, Marker

    Pattern of immunohistochemical staining for cleaved caspase-3 in the brains of fatal CM cases. A strong neuronal immunoreactivity for cleaved caspase-3 was observed in the neurons (arrows) (A) and glial cells (arrowheads) (B and C) compared with the neurons (D) and glial cells (E) of the normal brain. Scale bars = 20 μm.

    Journal: Malaria Journal

    Article Title: Nuclear factor kappa B modulates apoptosis in the brain endothelial cells and intravascular leukocytes of fatal cerebral malaria

    doi: 10.1186/1475-2875-12-260

    Figure Lengend Snippet: Pattern of immunohistochemical staining for cleaved caspase-3 in the brains of fatal CM cases. A strong neuronal immunoreactivity for cleaved caspase-3 was observed in the neurons (arrows) (A) and glial cells (arrowheads) (B and C) compared with the neurons (D) and glial cells (E) of the normal brain. Scale bars = 20 μm.

    Article Snippet: During the ensuing days, sections were washed three times with PBS and incubated with secondary antibody for 30 min at room temperature and reacted with avidin-biotin complex (ABC) conjugated with horseradish peroxidase (HRP) (for NF-κB p65) or alkaline phosphatase (AP) (for cleaved caspase-3) (Vectastain ABC kit, Vector Laboratories, CA, USA), according to the manufacturer’s instructions.

    Techniques: Immunohistochemistry, Staining

    Immunohistochemical staining for cleaved caspase-3 in the blood vessel of fatal CM brain. A micrograph to demonstrate strong positive staining for cleaved caspase-3 in the ECs (arrows) sequestered with PRBCs and leukocytes (arrowheads) inside the vascular lumen. VL = vascular lumen. Scale bars = 20 μm.

    Journal: Malaria Journal

    Article Title: Nuclear factor kappa B modulates apoptosis in the brain endothelial cells and intravascular leukocytes of fatal cerebral malaria

    doi: 10.1186/1475-2875-12-260

    Figure Lengend Snippet: Immunohistochemical staining for cleaved caspase-3 in the blood vessel of fatal CM brain. A micrograph to demonstrate strong positive staining for cleaved caspase-3 in the ECs (arrows) sequestered with PRBCs and leukocytes (arrowheads) inside the vascular lumen. VL = vascular lumen. Scale bars = 20 μm.

    Article Snippet: During the ensuing days, sections were washed three times with PBS and incubated with secondary antibody for 30 min at room temperature and reacted with avidin-biotin complex (ABC) conjugated with horseradish peroxidase (HRP) (for NF-κB p65) or alkaline phosphatase (AP) (for cleaved caspase-3) (Vectastain ABC kit, Vector Laboratories, CA, USA), according to the manufacturer’s instructions.

    Techniques: Immunohistochemistry, Staining

    Quantification of cleaved caspase-3 expression in neurons (A) and glial cells (B) in the brain of the control, NCM, and CM groups. a Significance of p

    Journal: Malaria Journal

    Article Title: Nuclear factor kappa B modulates apoptosis in the brain endothelial cells and intravascular leukocytes of fatal cerebral malaria

    doi: 10.1186/1475-2875-12-260

    Figure Lengend Snippet: Quantification of cleaved caspase-3 expression in neurons (A) and glial cells (B) in the brain of the control, NCM, and CM groups. a Significance of p

    Article Snippet: During the ensuing days, sections were washed three times with PBS and incubated with secondary antibody for 30 min at room temperature and reacted with avidin-biotin complex (ABC) conjugated with horseradish peroxidase (HRP) (for NF-κB p65) or alkaline phosphatase (AP) (for cleaved caspase-3) (Vectastain ABC kit, Vector Laboratories, CA, USA), according to the manufacturer’s instructions.

    Techniques: Expressing

    Quantification of cleaved caspase-3 in vascular ECs and intravascular leukocytes in the brain of control, NCM and CM groups. Data were analysed as apoptotic index in the vascular ECs (A) and intravascular leukocytes (B) . a Significance of p

    Journal: Malaria Journal

    Article Title: Nuclear factor kappa B modulates apoptosis in the brain endothelial cells and intravascular leukocytes of fatal cerebral malaria

    doi: 10.1186/1475-2875-12-260

    Figure Lengend Snippet: Quantification of cleaved caspase-3 in vascular ECs and intravascular leukocytes in the brain of control, NCM and CM groups. Data were analysed as apoptotic index in the vascular ECs (A) and intravascular leukocytes (B) . a Significance of p

    Article Snippet: During the ensuing days, sections were washed three times with PBS and incubated with secondary antibody for 30 min at room temperature and reacted with avidin-biotin complex (ABC) conjugated with horseradish peroxidase (HRP) (for NF-κB p65) or alkaline phosphatase (AP) (for cleaved caspase-3) (Vectastain ABC kit, Vector Laboratories, CA, USA), according to the manufacturer’s instructions.

    Techniques:

    Immunohistochemical staining for cleaved caspase-3 in the area of haemorrhage in the brains of fatal CM cases. (A) Representative photograph of area of congestion and ring haemorrhage is shown with H E stain. (B) Many glial cells located around and within the area of haemorrhage show strong positive staining for cleaved caspase-3 (arrows). Scale bars = 50 μm.

    Journal: Malaria Journal

    Article Title: Nuclear factor kappa B modulates apoptosis in the brain endothelial cells and intravascular leukocytes of fatal cerebral malaria

    doi: 10.1186/1475-2875-12-260

    Figure Lengend Snippet: Immunohistochemical staining for cleaved caspase-3 in the area of haemorrhage in the brains of fatal CM cases. (A) Representative photograph of area of congestion and ring haemorrhage is shown with H E stain. (B) Many glial cells located around and within the area of haemorrhage show strong positive staining for cleaved caspase-3 (arrows). Scale bars = 50 μm.

    Article Snippet: During the ensuing days, sections were washed three times with PBS and incubated with secondary antibody for 30 min at room temperature and reacted with avidin-biotin complex (ABC) conjugated with horseradish peroxidase (HRP) (for NF-κB p65) or alkaline phosphatase (AP) (for cleaved caspase-3) (Vectastain ABC kit, Vector Laboratories, CA, USA), according to the manufacturer’s instructions.

    Techniques: Immunohistochemistry, Staining

    Broader cross-reactive antibody responses are induced by prime-boost-boost vaccination with Addavax adjuvanted rHA as compared to infection with A/Hong Kong/1/1968 (A/HK68 H3N2) influenza virus. ( A ). The rHA specific IgG concentrations of heterosubtypic and stalk-specific antibodies reaction induced by vaccination with adjuvant alone control, rHA combined with Addavax adjuvant (Vax I, II and III), rHA without adjuvant (Vax Ø), or infection (Inf) with A/HongKong/1/68 (H3N2) virus, were determined using a 29-panel mPlex-Flu assay, and represented as a heatmap. ( B ). The selected antibody responses against influenza virus HA of A/HK68 were plotted over time. The antibody concentrations (ng/mL) of individual HA were calculated from the standard curves generated in same assay. Serum of individual mice were harvested before priming, as well as before and post-boost 21 day. The specific antibody concentration of IgG is shown as the mean ± standard deviations (STD) of the mean of individual mice (n = 6). The two-way analysis of variance (ANOVA) statistical analysis was conducted including experimental group and time as factors. ** p

    Journal: PLoS ONE

    Article Title: Broad cross-reactive IgG responses elicited by adjuvanted vaccination with recombinant influenza hemagglutinin (rHA) in ferrets and mice

    doi: 10.1371/journal.pone.0193680

    Figure Lengend Snippet: Broader cross-reactive antibody responses are induced by prime-boost-boost vaccination with Addavax adjuvanted rHA as compared to infection with A/Hong Kong/1/1968 (A/HK68 H3N2) influenza virus. ( A ). The rHA specific IgG concentrations of heterosubtypic and stalk-specific antibodies reaction induced by vaccination with adjuvant alone control, rHA combined with Addavax adjuvant (Vax I, II and III), rHA without adjuvant (Vax Ø), or infection (Inf) with A/HongKong/1/68 (H3N2) virus, were determined using a 29-panel mPlex-Flu assay, and represented as a heatmap. ( B ). The selected antibody responses against influenza virus HA of A/HK68 were plotted over time. The antibody concentrations (ng/mL) of individual HA were calculated from the standard curves generated in same assay. Serum of individual mice were harvested before priming, as well as before and post-boost 21 day. The specific antibody concentration of IgG is shown as the mean ± standard deviations (STD) of the mean of individual mice (n = 6). The two-way analysis of variance (ANOVA) statistical analysis was conducted including experimental group and time as factors. ** p

    Article Snippet: Plates were then incubated overnight at 4°C, washed six times with PBST, then IgG specific spots were developed using the Alkaline Phosphate Substrate Kit III (Vector Laboratories, Burlingame, Ca).

    Techniques: Infection, Generated, Mouse Assay, Concentration Assay