Vimentin vim

Immunofluorescence was performed to assess the primary antibody of VEGF-A and KI67 according to the manufacturer’s procedures. The sections were treated with primary antibodies, namely, VEGF-A (1:200, Abcam, Cambridge, United Kingdom) and KI67 (1:200, Abcam, Cambridge, United Kingdom), overnight. Then, the sections were incubated with fluorogenic secondary antibodies for 1 h at 37°C. Finally, the sections were washed with PBS and incubated with DAPI for 10 min. For immunohistochemistry, the sections were treated with a primary antibody, namely, Vimentin (VIM; 1:200, Abcam, Cambridge, United Kingdom), overnight at 4°C and then incubated with horseradish-peroxidase-conjugated secondary antibody for another 2 h at 37°C. Then, the sections were developed using 3,3′-diaminobenzidine. All sections were observed using a fluorescence microscope (Eclipse 80i, Nikon, Japan).

BRCA1^f/f and Tg (MMTV-Cre) 4Mam mice were obtained from the NCI Mouse Repository and JAX lab, respectively [46 (link), 47 (link)]. The generation of p16^−/−, p18^−/−, BRCA1^+/−, BRCA1^MGKO(BRCA1^f/f;MMTV-Cre or BRCA1^f/−;MMTV-Cre) mice was described previously [31 (link), 48 (link)–50 (link)]. The Institutional Animal Care and Use Committee at the University of Miami and Shenzhen University approved all animal procedures. Histopathology and immunohistochemistry (IHC) were performed as described previously [11 (link), 28 (link), 31 (link)]. The primary antibodies used were Ck14, cleaved caspase 3 (Thermal Scientific), PDGFRβ, phosphorylated PKCα (p-PKCα), phosphorylated FRA1 (p-FRA1), FRA1 (Cell signaling), ERα, BRCA1 (Santa Cruz), E-cadherin (E-cad), PKCα (BD Biosciences), and Vimentin (Vim) (Abcam). Immunocomplexes were detected by using the Vectastain ABC DAB kit according to the manufacturer’s instructions (Vector Laboratories) or by using FITC- or rhodamine-conjugated secondary antibodies (Jackson Immunoresearch).

The presence of surface marker antigens was detected using antibodies CD90/THY1 (Stem Cell Technologies, Vancouver, BC, Canada), CD133/PROM1, CD45 (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany), CD44 (Abcam, Cambridge, UK), and STRO-1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). After detachment, at least two million cells per mL were incubated with specific first antibodies for 60 minutes on ice in the dark. After two washings with PBS containing 0.5% bovine serum albumin (BSA), when necessary, the cells were incubated with fluorescence-conjugated secondary antibody for 60 minutes on ice in the dark. Flow cytometric analysis was performed immediately in a FACSCalibur flow cytometer (Becton Dickinson, NJ, USA). Ten thousands events were analysed per sample.
The presence and localization of the proteins CD90, CD44, Vimentin/Vim (Abcam), and alpha smooth muscle actin/αSMA (Dako, Glostrup, Denmark) were visualized with immunofluorescence using fluorescence microscope Leica DM2000 (Leica Camera AG, Solms, Germany). The nucleus of the cells was stained with Hoechst 33342 dye (Sigma-Aldrich).