Cy3 conjugated goat anti rabbit antibody

Cells were seeded on chamber slides (LabTek, ThermoFisher SCIENTIFIC, Lausanne, Switzerland) in growth medium for 1 day. The next day, cells were treated for 7 days, as mentioned above. The indirect immunostainings for cells were performed at 37 °C with overnight incubation using the primary antibodies anti-PSMA/FOLH1 (clone 460420, 1:100, R&D Systems, Zug, Switzerland) and AR (clone D6F11, 1:500, CellSignaling, Leiden, The Netherlands). The slides were incubated with secondary antibodies: goat anti-mouse FITC (1:500, Vector Laboratories) or Cy3-conjugated goat anti-rabbit antibody (Sigma, 1:1000) at room temperature for 1 h. After counter-staining with DAPI (4′, 6-diamidino-2-phenylindole, Sigma, 1:200), the slides were analyzed by Thunder imaging system (Leica DMi₈ Thunder inverted microscope, Mannheim, Germany). Fluorescence intensity was measured using Image J (1.4, NIH, USA).

Dulbecco’s modified Eagle’s medium (DMEM)/F12 culture medium was purchased from Life Technologies (Gaithersburg, MD, USA). Fetal bovine serum (FBS) and B27 supplement were purchased from Gibco-BRL (Grand Island, NY, USA). Basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) were obtained from PeproTech (Rocky Hill, NJ, USA). Normal goat serum was provided by Wuhan Boshide Biotechnology Co., Ltd. (Wuhan, China). Poly-L-lysine, 4′,6-diamidino-2-phenylindole (DAPI), fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit antibody and Cy3-conjugated goat anti-rabbit antibody were provided by Sigma (St. Louis, MO, USA). Rabbit anti-mouse CD133 monoclonal antibody and glial fibrillary acidic protein (GFAP) polyclonal antibody were purchased from Abcam (Cambridge, MA, USA) and Zhongshan Jinqiao Biotechnology, Ltd., (Beijing, China), respectively. Anti-caspase-3 antibody (rabbit polyclonal against mouse, rat or human) and anti-β-actin antibody were obtained from Abcam Inc. (Cambridge, MA, USA). Cell Counting kit-8 (CCK-8) was purchased from Dojindo Laboratories (Tokyo, Japan).