Mouse monoclonal anti mvh

The double staining of MVH and STELLA was conducted as follows. FGSCs were fixed with 4% PFA for 30 min at room temperature. They were then incubated in blocking solution containing 10% normal goat serum for 60 min, and incubated with mouse monoclonal anti-MVH (1:100, Abcam, Cambridge, UK) overnight at 4℃. Cells were washed once with PBS and permeabilized with 0.5% Triton X-100 for 30 min. Next, the cells were incubated with rabbit polyclonal anti-STELLA (1:200, Abcam, Cambridge, UK) overnight at 4℃. After washing with PBS three times, the cells were incubated with the secondary antibody for 60 min, washed, and then incubated with DAPI for 10 min. Images were acquired using a Leica digital camera.
For OCT4 staining, before incubation in blocking buffer, cells were permeabilized with 0.5% Triton X-100 for 30 min and then washed with PBS three times. The remaining steps were the same as for the above double staining. The primary antibody was rabbit polyclonal anti-OCT4 (1:100, Santa Cruz, CA, USA).

The immunofluorescence procedure was performed as described previously with minor modification [30 (link)]. Cultured cells were washed twice with phosphate-buffered saline (PBS) and then fixed in 4% paraformaldehyde for 15 min. After that the cells were rinsed twice and incubated with PBS containing 0.1% (v/v) Triton X-100 for 15 min. Fixed cells were blocked by 10% normal horse serum in PBS for 10 min at room temperature. The cells were then incubated with primary antibodies: mouse monoclonal anti-NESTIN and rabbit polyclonal anti-SOX2 (1:180 dilution; Abcam), mouse polyclonal anti-TUJ-1 and rabbit polyclonal anti-GFAP (1:150 dilution; Abcam), mouse monoclonal anti-MVH (1:500 dilution; Abcam), mouse monoclonal anti-PLZF (1:100 dilution; Santa Cruz Biotechnology). After 1 h of incubation at room temperature with the primary antibodies, the cells were rinsed twice in PBS and then incubated in the dark with a fluorescein isothiocyanate-conjugated secondary antibody, either goat antirabbit IgG or goat anti-mouse IgG (1:200 dilution; Proteintech, Chicago, IL, USA) for 60 min at 37 °C. This was followed by rinsing and staining of the nucleus with 4′,6-diamidino-2-phenylindole (DAPI) (1:1000 dilution; Sigma)-containing PBS for 10 min at room temperature. After washing twice with PBS, the cells were examined in fresh PBS under an inverted fluorescence microscope (Leica).