With the approval of our institutional review board, formalin-fixed paraffin-embedded (FFPE) blocks from breast tumor specimens were retrieved and reviewed to confirm the presence of normal ductal epithelium, ductal carcinoma in situ, and invasive ductal carcinoma. Breast and ovarian cancer cell lines with known PTEN status served as positive and negative controls. The PTEN status in cell lines was confirmed by Western blotting using the anti-PTEN antibody (mouse anti-human, clone 6H2.1, Dako). The breast cancer cell line MDA-MB-468 and ovarian cancer cell line IGROV-1 had no PTEN expression, whereas PTEN wild-type MCF7 and SKOV3 cell lines showed positive PTEN protein expression. Cell pellets were processed and embedded in paraffin using standard techniques. The samples and controls were sectioned (4 mm) and stained on the Dako Autostainer Plus (Dako USA, Carpinteria, CA).
Before immunostaining, the slides were heated (56°C) for 3 hours in a drying oven and then deparaffinized (xylene), washed with alcohol (100% and 95%), and rehydrated in deionized water. Antigen retrieval was performed as follows: the slides were incubated at 98°C for 20 minutes in Target Retrieval Solution pH 9 (Tris/ethylene diamine tetra-acetate buffer, pH 9, Dako Cytomation), then allowed to cool to room temperature before rinsing with Tris-buffered saline wash buffer (Dako). Endogenous peroxidase activity was blocked by incubating the slides for 5 minutes in 0.03% hydrogen peroxide (EnVision/HRP, Dako). After rinsing in wash buffer, the sections were incubated for 30 minutes at room temperature with the monoclonal mouse anti-human PTEN antibody (dilution 1:100, clone 6H2.1, Dako) in Tris-HCl buffer antibody diluent (Dako). Slides were rinsed in wash buffer and incubated for 30 minutes with peroxidase-labeled polymer conjugated to goat anti-mouse immunoglobulins (EnVision/HRP, Dako). The chromogenic reaction was carried out with 3,3′-diaminobenzidine chromogen solution for 10 minutes, resulting in the expected brown-colored signal. Finally, after rinsing with deionized water, the slides were counterstained with hematoxylin, dehydrated, mounted with toluene-based mounting medium (Thermo Scientific Richard-Allan) and coverslipped ().
PTEN Immunohistochemistry Staining Protocol