All lanes : Anti-Hsp27 antibody (ab5579) at 1/2000 dilution

Lane 1 : MCF7 (human breast adenocarcinoma cell line) whole cell lysate
Lane 2 : MDA-MB-231 (human breast adenocarcinoma cell line) whole cell lysate
Lane 3 : PC3 (human prostate adenocarcinoma cell line) whole cell lysate
Lane 4 : DU 145 (human prostate carcinoma cell line) whole cell lysate
Lane 5 : HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 6 : LNCaP (human prostate cancer cell line) whole cell lysate
Lane 7 : HT1080 (human fibrosarcoma cell line) whole cell lysate

Lysates/proteins at 30 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP at 1/4000 dilution

Immunofluorescence analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Hsp27 (green) with ab5579 at 1/200 dilution, followed by DyLight 488-conjugated secondary antibody. F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue). Cells were fixed with formaldehyde and incubated with the primary antibody overnight at 4°C. 60X magnification. Right - negative control.

Immunohistochemical analysis of both normal and cancer biopsies of deparaffinized human skeletal muscle tissue labeling Hsp27 with ab5579 at 1/20 dilution or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH 6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature.

Immunofluorescence analysis of MCF7 (Human breast adenocarcinoma cell line) cells labeling Hsp27 (green) with ab5579 at 1/200 dilution, followed by DyLight 488-conjugated secondary antibody. F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue). Cells were fixed with formaldehyde and incubated with the primary antibody overnight at 4°C. 60X magnification. Right - negative control.

Immunohistochemical analysis of both normal and cancer biopsies of deparaffinized human breast carcinoma tissue labeling Hsp27 with ab5579 at 1/100 dilution or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. 

Immunofluorescence analysis of C6 (Rat glial tumor cell line) cells labeling Hsp27 (green) with ab5579 at 1/100 dilution, followed by DyLight 488-conjugated secondary antibody. F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue). Cells were fixed with formaldehyde and incubated with the primary antibody overnight at 4°C. 60X magnification. Right - negative control.

All lanes : Anti-Hsp27 antibody (ab5579) at 1/1000 dilution

Lane 1 : HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate
Lane 2 : HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 3 : K562 (human chronic myelogenous leukemia lymphoblast cell line ) whole cell lysate
Lane 4 : A431 (human epidermoid carcinoma cell line) whole cell lysate
Lane 5 : HepG2 (human liver hepatocellular carcinoma cell line) whole cell lysate
Lane 6 : COS-7 (african green monkey kidney fibroblast-like cell line) whole cell lysate
Lane 7 : NIH/3T3 (mouse embryonic fibroblast cell line) whole cell lysate

Lysates/proteins at 50 µg per lane.

Secondary
All lanes : Goat anti-rabbit IgG-HRP secondary antibody at 1/20000 dilution

HeLa (human epithelial cell line from cervix adenocarcinoma) 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 1/250 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 minute