Immunocytochemistry/Immunofluorescence analysis of HSP90 alpha (green) in HeLa (Human epithelial cell line from cervix adenocarcinoma) cells and NIH/3T3 (Mouse embryo fibroblast cell line) cells. Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% BSA for 15 minutes at room temperature. Cells were incubated with ab2928 at a dilution of 1:100 for at least 1 hour at room temperature, washed with PBS, and incubated with a DyLight 488 goat-anti-rabbit IgG secondary antibody (1:400) for 30 minutes at room temperature. Nuclei (blue) were stained with Hoechst 33342 dye. Images were taken at 20X magnification.

Western blot analysis of HSP90 alpha was performed by loading samples onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked with 5% BSA/TBST for at least 1 hour. The membrane was probed with ab2928 overnight at 4°C on a rocking platform, washed in TBS-0.1%Tween 20, and probed with a secondary antibody for at least 1 hour. Chemiluminescent detection was performed.

ab2928 labelling Hsp90 alpha in Human colon adenocarcinoma tissue sections by Immunohistochemistry (formalin/PFA-fixed paraffin-embedded sections). To expose target proteins, heat-induced epitope retrieval was performed using 10mM sodium citrate (pH 6.0) buffer for 20 minutes at 95ºC. Tissues were blocked in 3% BSA in PBST for 30 minutes at room temperature. Tissue sections were incubated with the primary antibody (1:100) for 1 hour. A HRP-conjugated goat anti-rabbit IgG (1:250) was used as the secondary antibody, followed by colorimetric detection using Metal Enhanced DAB Substrate Kit. Tissues were counterstained with hematoxylin and prepped for mouting. Images were taken at 40X magnification.

Immunoprecipitation of HSP90 alpha was performed on HeLa (Human epithelial cell line from cervix adenocarcinoma) cells. Antigen-antibody complexes formed by incubating 500ug whole cell lysate with 2ug of ab2928 overnight on a rocking platform at 4°C. The immune complexes were captured on 50ul Protein A/G Plus Agarose, washed extensively, and eluted with buffer. Samples were then resolved on a 4-20% Tris-HCl polyacrylamide gel, transferred to a PVDF membrane and blocked with 5% BSA/TBST for at least 1 hour. The membrane was probed with ab2928 at a dilution of 1:1000 overnight rotating at 4°C. The membrane was washed in TBST, and probed with detection reagent at a dilution of 1:1000 for at least 1 hour. Chemiluminescent detection was performed.

Immunohistochemistry was performed on both normal and cancer biopsies of deparaffinized human breast carcinoma tissues. 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. Tissues were then probed at a dilution of 1:50 with a rabbit polyclonal antibody recognizing Heat Shock Protein 90 (86) ab2928 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.

Immunohistochemistry was performed on both normal and cancer biopsies of deparaffinized human tonsil tissue. 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. Tissues were then probed at a dilution of 1:200 with a rabbit polyclonal antibody recognizing Heat Shock Protein 90 (86) ab2928 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.

Immunohistochemistry was performed on both normal and cancer biopsies of deparaffinized human kidney tissue. 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. Tissues were then probed at a dilution of 1:100 with a rabbit polyclonal antibody recognizing Heat Shock Protein 90 (86) ab2928 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.