Immunocytochemical Staining of Cell Markers

Immunocytochemical staining was conducted on cell-bearing coverslips by the method described elsewhere [15 (link)]. The antibodies used were: Cyclin D1 (Bioss Inc., Beijing, China; 1:100), Tg (Bioss. Inc., Beijing, China; 1:200), E-cadherin (Bioss. Inc., Beijing, China; 1:100), RAR-β (Bioss. Inc., Beijing, China; 1:150), and PPAR-β/δ (Bioss. Inc., Beijing, China; 1:200). Cells incubated in 0.2% DMSO-containing medium were used as a control. The color reaction was performed by using 3,3′-diaminobenzidine tetrahydrochloride (DAB) after the binding of the primary antibody (Vector Laboratories, Burlingame, CA, USA). For double immunofluorescent staining (IF), mouse anti-CRABP2 and rabbit anti-FABP5 were employed in the working concentrations of 1:120. Briefly, the cell-bearing coverslips were washed with phosphate-buffered solution (PBS, pH 7.4), incubated in 3% H₂O₂ for 10 min and then with anti-CRABP2 (1:120; Proteintech, Chicago, IL, USA) and anti-FABP5 (1:120; Proteintech, Chicago, IL, USA) at 4 °C for one night in a humid chamber. Finally, the coverslips were co-incubated with FITC-conjugated goat anti-mouse IgG and PE-conjugated goat anti-rabbit IgG (both 1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 37 °C for 60 min in the dark, sealed with fluorescence mounting medium, and observed and imaged under a fluorescence microscope (BX53F, Olympus, Tokyo, Japan).