Gata4

Testis samples from 3-month-old goats were fixed with 4% paraformaldehyde. The samples were cryo-embedded in optimum cutting temperature (OCT) compound and then cut into 7 μm thick sections. MT1 (Santa Cruz Biotechnology sc-13180), MT2 (Santa Cruz Biotechnology sc-13177), RORα (Abcam ab60134) and GATA-4 (Santa Cruz Biotechnology sc-1237) distributions were examined using immunohistochemistry. Briefly, after washing three times with PBS, the slides were incubated with PBS containing 1% bovine serum albumin (BSA) for 1 h at room temperature. Primary antibodies directed against MT1 (final concentration 1:200), MT2 (final concentration 1:200), GATA-4 (final concentration 1:200), and RORα (final concentration 1:200) were added to the solution. After 4 hours of incubation, the secondary antibody was applied for 1 h. Staining was visualized using a 3,3’-diaminobenzidine (DAB) substrate kit.

Cells were fixed in 4% (wt/vol) paraformaldehyde for 10 min, permeabilized with 0.2% Triton X‐100 for 5 min and blocked in 3% (vol/vol) goat serum (Dako) or donkey serum (Sigma) for 45 min. They were then incubated in primary antibodies for 45 min followed by secondary antibodies for 30 min (Alexa Fluor dyes, 1:1000, Invitrogen). All antibodies were diluted in the blocking buffer. Nuclei were counterstained with DAPI (Sigma) for 5 min and coverslips were mounted on slides with FluorSave (Merck). All procedures were performed at room temperature. Primary antibodies used in this study were Vimentin (1:100, Millipore), NFIA (1:250, abcam), GFAP (1:500, Dako), GFAP (1:500, Sigma), S100B (1:500, Dako), βIII‐tubulin (1:1000, Sigma), TDP‐43 (1:250, Abnova), NANOG (1:250, R&D Systems), SOX2 (1:250, Millipore), TRA‐1‐60 (1:250, Santa Cruz), OCT3/4 (1:250, Santa Cruz), SOX1 (1:100, R&D Systems), Nestin (1:1000, Millipore), Brachyury (1:100, R&D Systems), EOMES (1:600, abcam), FOXA2 (1:100, R&D Systems), GATA‐4 (1:100, Santa Cruz), SMI32 (1:250, Covance), and Caspase‐3 (1:500, Abcam).
Fluorescent imaging was performed on fields of view containing uniform DAPI staining using either an Axio Observer.Z1 (Zeiss) epifluorescence microscope or an LSM710 confocal microscope (Carl Zeiss). Images were processed and blindly analyzed by using the ImageJ64 (v 1.47) software.

For immunofluorescence staining, cells were washed with PBS and fixed with 4% paraformaldehyde for 10 min, permeabilized with 1%Triton X-100 (Sigma) for 1 h, blocked with 5%BSA (Sigma) for 1 h at room temperature, then incubated with primary antibodies at 4 °C overnight and then incubated with secondary antibodies for 1 h at room temperature. The primary antibodies included Gata4 (Santa Cruz Biotechnology, Dallas, TX, USA), Gata6 (Cell Signaling Technology, Danvers, MA, USA), Sox17 (Cell Signaling Technology), Sall4 (Santa Cruz Biotechnology), Oct4 (Santa Cruz Biotechnology), Sox2 (Calbiochem, Billerica, MA, USA), Nanog (Santa Cruz Biotechnology), Cdx2 (Bio Genex, San Ramon, CA, USA), Sparc (Santa Cruz Biotechnology), Cdh1 (Abcam, Waltham, MA, USA), Desmin (Millipore), Cytokeratin (Millipore), and βIII-Tubulin (Abcam). Secondary antibodies include goat anti-mouse IgG Alexa Fluor 546 (Thermo Fisher Scientific, Waltham, MA, USA), goat anti-mouse IgG Alexa Fluor 488 (Thermo Fisher Scientific), goat anti-rabbit IgG Alexa Fluor 546 (Thermo Fisher Scientific), and goat anti-rabbit IgG Alexa Fluor 488 (Thermo Fisher Scientific). The nuclei were stained with DAPI (Sigma).