Application Notes: Western blot, 0.1-0.5µg/ml Immunohistochemistry (Paraffin-embedded Section), 0.5-1µg/ml Immunocytochemistry/Immunofluorescence, 2µg/ml Flow Cytometry (Fixed), 1-3µg/1x106 cells. Add 0.2ml of distilled water will yield a concentration of 500µg/ml
Flow Cytometry analysis of A549 cells using anti-HSP27 antibody. Overlay histogram showing A549 cells (Blue line). To facilitate intracellular staining, cells were fixed with 4% paraformaldehyde and permeabilized with permeabilization buffer. The cells were blocked with 10% normal goat serum. And then incubated with mouse anti-HSP27 Antibody (1 µg/1x10 6 cells) for 30 min at 20C. DyLight488 conjugated goat anti-mouse IgG (5-10 µg/1x10 6 cells) was used as secondary antibody for 30 minutes at 20C. Isotype control antibody (Green line) was mouse IgG (1 µg/1x10 6) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.
Flow Cytometry analysis of U20S cells using anti-HSP27 antibody. Overlay histogram showing U20S cells (Blue line). To facilitate intracellular staining, cells were fixed with 4% paraformaldehyde and permeabilized with permeabilization buffer. The cells were blocked with 10% normal goat serum. And then incubated with mouse anti-HSP27 Antibody (1 µg/1x10 6 cells) for 30 min at 20C. DyLight488 conjugated goat anti-mouse IgG (5-10 µg/1x10 6 cells) was used as secondary antibody for 30 minutes at 20C. Isotype control antibody (Green line) was mouse IgG (1 µg/1x10 6) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.
IF analysis of HSP27 using anti-HSP27 antibody. HSP27 was detected in immunocytochemical section of U20S cell. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 2 µg/mL mouse anti-HSP27 Antibody overnight at 4C. DyLight488 Conjugated Goat Anti-mouse IgG was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.
IHC analysis of HSP27 using anti-HSP27 antibody. HSP27 was detected in paraffin-embedded section of human lung cancer tissues. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1 µg/ml mouse anti-HSP27 Antibody overnight at 4C. Biotinylated goat anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) with DAB as the chromogen.
IHC analysis of HSP27 using anti-HSP27 antibody. HSP27 was detected in paraffin-embedded section of human oesophagus squama cancer tissues. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1 µg/ml mouse anti-HSP27 Antibody overnight at 4C. Biotinylated goat anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) with DAB as the chromogen.
IHC analysis of HSP27 using anti-HSP27 antibody. HSP27 was detected in paraffin-embedded section of human placenta tissues. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1 µg/ml mouse anti-HSP27 Antibody overnight at 4C. Biotinylated goat anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) with DAB as the chromogen.
Western blot analysis of HSP27 using anti-HSP27 antibody. Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of
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