Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) was performed on human uterus tissue. Antigen retrieval was performed using 10mM sodium citrate followed by microwave treatment for 8-15 minutes. Endogenous peroxidases were blocked in 3% H202-methanol for 15 minutes and tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Cells were incubated with ab2765 (1:20) overnight in a humidified chamber. Tissues were washed in PBST and detection was performed using a secondary antibody conjugated to HRP. DAB staining buffer was applied and tissues were counterstained with hematoxylin and prepped for mounting. Images were taken at 40X magnification.

Immunocytochemistry/ Immunofluorescence analysis of T47D cells untreated (left) or stimulated with 100nm promegestone for 1 hour (right), labeling Progesterone Receptor with ab2765 (green). The cells were fixed with formalin for 15 minutes, permeabilized with 0.1% Triton X-100 in TBS for 10 minutes, and blocked with 3% Blocker BSA for 15 minutes at room temperature. Cells were stained with Anti-Progesterone Receptor antibody [Alpha PR6] - ChIP Grade (ab2765) at a dilution of 1/100 for 1 hour at 37C, and then incubated with a Alexa Fluor 488 goat anti-mouse IgG secondary antibody at a dilution of 1/1000 for 30 minutes at room temperature (both panels, green). Nuclei (both panels, blue) were stained with Hoechst 33342 dye.

All lanes : Anti-Progesterone Receptor antibody [Alpha PR6] (ab2765) at 1 µg/ml

Lane 1 : T47D cell lysate untreated (-)
Lane 2 : T47D cell lysate stimulated (+) with 100 nm promegestone for 1 hour

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Mouse IgG-HRP at 1/2000 dilution

Predicted band size: 99 kDa

Overlay histogram showing T47D cells stained with ab2765 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab2765, 0.5µg/1x10⁶ cells) 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 IgG2a [ICIGG2A] (ab91361, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.