Goat anti rat igg

Cells were fixed with 4% paraformaldehyde for 30 min at room temperature and permeabilized with 0.5% Triton X-100 (Solarbio, Beijing, China vT8200) for 10 min. The samples were then blocked with blocking buffer (PBS containing 10% fetal calf serum [FBS]) (Gibco, New York, USA, 10099141) at 37 °C for 2 h or 4 °C overnight. Primary antibodies diluted in blocking buffer were used for the samples and incubated at 37 °C for 2 h or 4 °C overnight. The samples were washed three times with PBS containing 0.1% Tween 20 (Solarbio, Beijing, China, T8220) followed by incubation with fluorescence-conjugated secondary antibodies diluted in blocking buffer for 2 h at 37 °C. The samples were washed three times with PBS containing 0.1% Tween 20. Nuclei were counterstained with DAPI (Beyotime, Beijing, China, C1002). Images were obtained using a confocal microscope (Olympus, Tokyo, Japan, FV1200). Primary antibodies included MVH (Abcam, Cambridge, UK, ab13840, 1:100) and CKIT (Invitrogen, California, USA, 14-1172-81, 1:100). Secondary antibodies included goat anti-rat IgG (Proteintech, Chicago, USA, SA00003-11, 1:1000, FITC labeled) and goat anti-mouse IgG (Proteintech, Chicago, USA, SA00003-12, 1:1000, TRITC labeled). The directly labeled antibody was SSEA-1 (Biotechne, Minnesota, USA, IC2155T, 1:1000). PGCs were used as a positive control, and iPSCs were employed as a negative control.

Dulbecco’s modified eagle medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco (Carlsbad, CA, U.S.A.). Human stem cell factor, basic fibroblast growth factor, retinoic acid (RA), and murine leukemia inhibitory factor were acquired from Sigma-Aldrich (Saint Louis, MO, U.S.A.). TRNzol, FastQuant-RT kit, and SuperReal premix color kit were obtained from TIANGEN (Beijing, China). Fugen was from Promega (Madison, WI, U.S.A.). Antibodies specific to the following proteins were integrinα6 (Millipore, temecula, CA, U.S.A.; dilution ratio 1:100), integrin β1 (Millipore, temecula, CA, U.S.A.; no. MAB1378; dilution ratio 1:100), goat anti-Rat IgG (Proteintech, Chicago, Illinois U.S.A.; no. SA00003-11; [FITC] labeled; dilution ratio, 1:100), and goat anti-rabbit IgG (Proteintech, Chicago, Illinois, U.S.A.; no. SA00008-9; [PE] labeled; dilution ratio, 1:100).