Anti-Hsp27 抗体 [G3.1] (ab2790)

Immunocytochemistry/ Immunofluorescence analysis of non small cell lung carcinoma NCI-H1299 cells labeling Hsp27 with ab2790 at 1/300 dilution. The cells were fixed with formaldehyde and permeabilised with 0.2% TritonX-100. The cells were blocked with 1.5% serum for 10 minutes at 25°C, followed by incubation with Anti-Hsp27 antibody [G3.1] (ab2790) in 1x HBSS + 0.02% TritonX-100 + 1.5% FBS for 3 hours at 25°C. A polyclonal goat anti-mouse IgG Alexa Fluor^® 488 was used as the secondary antibody at 1/1000 dilution. DAPI nuclear stainining.

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Immunohistochemistry of paraffin embedded human breast carcinoma with ab2790 labeling Hsp27 at 0.5μg/ml.

Immunohistochemistry of paraffin embedded human prostate carcinoma with ab2790 labeling Hsp27 at 0.5μg/ml.

Cells were fixed with 4% formaldehyde in PEM buffer. The coverslip was incubated in blocking buffer of 5% powdered milk in TBS-T plus 0.02% sodium azide for 1 hour at room temperature. Blocking buffer was removed and primary antibody was added at a dilution of 1/200 and incubated overnight at 4 degrees celsius. The coverslips were then washed 4-5 times with blocking buffer for 5 minutes. Secondary antibody, goat anti-rabbit Alexa 594 (ab150080), was added at a dilution of 1/1000 and incubated at room temperature for one hour. From this point on coverslips were covered with foil to protect them from light. They were washed 5 times with TBS-T and then one time with PEM, for 5 minutes each wash. The coverslips were fixed 10-30 minutes in 4% formaldehyde in PEM buffer, then washed 3 times with PEM buffer for 5 minutes. 0.1M ammonium chloride in PEM buffer was added for 10 minutes to quench auto-florescence, and then slips were washed 2 times for 5 minutes in PEM followed by 3 washes for 5 minutes in

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Overlay histogram showing HeLa cells (ab150035) stained with ab2790 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum (ab7481)  / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab2790, 1/100 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight^® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

ab2790 staining Hsp27 in A549 lung cancer cells by Flow Cytometry. Cells were harvested in trypsin, fixed with paraformaldehyde and permeabilized with 0.2% Triton X-100. The sample was incubated with the primary antibody (1/50 in 1x HBSS + 0.02% Triton X-100 + 1.5% FBS) for 3 hours at 25°C. An Alexa Fluor^® 488-conjugated goat anti-mouse IgG polyclonal (1/2000) was used as the secondary antibody. Gating Strategy: No gating.

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Flow Cytometry analysis of mouse lewis lung carcinoma cells labeling Hsp27 with ab2790. Cells were harvested in trypsin, then fixed with praformaldehyde and permeabilized with 0.2% Triton X-100. Followed by incubation with Anti-Hsp27 antibody [G3.1] (ab2790)  at 1/200 dilution in 1x HBSS + 0.02% Triton X-100 + 1.5% FBS for 3 hours at 25°C. A polyclonal goat anti-mouse IgG Alexa Fluor^® 488 secondary antibody was used at 1/2000.

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