Estrogen receptor

Western blot analysis was performed as previously described [25 (link)]. In brief, cultured cells were washed with ice-cold PBS and lysed in RIPA buffer at 4°C containing a cocktail of protease inhibitors (Sigma-Aldrich, P8340). Lysates were clarified by centrifugation. The supernatants were collected and total protein concentration was determined [28 (link)]. After SDS-PAGE, proteins were transferred onto nitrocellulose membranes for western blot analysis. Membranes were incubated with specific antibodies: CAIX (M75 monoclonal, gift from Dr. Egbert Oosterwijk), CAII (Novus, #NB600-919), CAXII (R&D, # AF2190), estrogen receptor (Santa Cruz, # sc-8002), E-cadherin (BD biosciences, #610181), GAPDH (Cell Signaling, #5174), and actin (Abcam, #ab8226) followed by incubation with horseradish peroxidase–conjugated secondary antibodies (Sigma-Aldrich). The secondary antibodies were detected by enhanced chemiluminescence (GE Healthcare Biosciences, #RPN2106).

Tissue block samples were fixed for 15 min at room temperature with 4% paraformaldehyde in PBS followed by 2–3 washes with PBS. Embedded cells within tissue blocks were permeabilized with PBS containing 0.4 M glycine and 0.3% Triton X-100, and nonspecific reactions were prevented with 1 h incubation in blocking solution containing 2% normal goat serum (DAKO, Glostrup, Denmark). Immunostaining of each tissue block was performed as described previously [25 (link)], using the primary anti-GFP (Invitrogen and Cat. No: V820-20), mCherry (TAKARA and Cat. No:632,524), estrogen receptor (Santa cruz biotechnology, Cat. No.: sc-8005), progesterone receptor (Santa cruz biotechnology, Cat. No.: sc-810), vimentin (BD biosciences, Cat. No.: 550,513), cytokeratin 14 (Santa cruz biotechnology, Cat. No.: sc-53,253), PECAM1 (R&D systems, Cat. No.: BBA7), von Willebrand factor (Abcam, Cat. No.: ab6994), CD11b (Abcam, Cat. No.: ab52478), CD68 (Santa cruz biotechnology, Cat. No.: sc-20,060), myogenin (Abcam, Cat. No.: ab1835), and myo D (Santa cruz biotechnology, Cat. No.: sc-377,460) antibodies. Protein expression patterns of embedded cells within each tissue blocks were examined under a fluorescence microscopy (Zeiss LSM 510 Meta).