Western blot of mouse HSP 86 using ab2927.

Immunocytochemistry/Immunofluorescence analysis of HSP90 beta shows staining in HepG2 cells. HSP 90 beta staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or ab2927 at a dilution of 1:100 over night at 4°C, washed with PBS and incubated with a DyLight-488 conjugated goat anti-rabbit secondary antibody. Images were taken at 60X magnification.

Immunocytochemistry/Immunofluorescence analysis of HSP90 beta shows staining in A2058 cells. HSP 90 beta staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or ab2927 at a dilution of 1:100 over night at 4°C, washed with PBS and incubated with a DyLight-488 conjugated goat anti-rabbit secondary antibody. Images were taken at 60X magnification.

Immunocytochemistry/Immunofluorescence analysis of HSP90 beta shows staining in U251 cells. HSP 90 beta staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or ab2927 at a dilution of 1:100 over night at 4°C, washed with PBS and incubated with a DyLight-488 conjugated goat anti-rabbit secondary antibody. Images were taken at 60X magnification.

Immunocytochemistry/Immunofluorescence analysis of HSP90 beta (green) in HeLa and NIH3T3 cells. Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature. Cells were blocked with 1% BSA for 15 minutes at room temperature. Cells were incubated with ab2927 at a dilution of 1:50 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.

Immunoprecipitation of Hsp90 was performed on HeLa cells. Antigen:antibody complexes were formed by incubating 500µg whole cell lysate with 2µg of Hsp90 polyclonal antibody (ab2927) overnight on a rocking platform at 4°C. Immune complexes were captured on 50µl Protein A/G Agarose washed extensively and eluted with Buffer. Samples were resolved on a 4-20% Tris-HCl polyacrylamide gel, transferred to a PVDF membraneand blocked with 5% BSA/TBST for at least 1 hour. The membrane was probed with a Hsp90 polyclonal antibody (ab2927) at a dilution of 1:1000 overnight rotating at 4°C, washed in TBSTand probed with HRP detection reagent at a dilution of 1:1000 for at least one hour. Chemiluminescent detection was performed.

Immunohistochemistry was performed on normal 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 1:100 with a rabbit polyclonal antibody recognizing Heat Shock Protein 84 ab2927 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 normal biopsies of deparaffinized Human placenta 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 1:20 with a rabbit polyclonal antibody recognizing Heat Shock Protein 84 ab2927 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 cancer biopsies of deparaffinized Human breast carcinoma 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 84 ab2927 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.