Meiotic Spread Preparation and Immunocytochemistry

Meiotic spread preparations were prepared as previously described (31 (link)). Alternatively, the cells were spread on the slides using a Cytospin (CELLSPIN, 521-1990, VWR) (17 (link)). Briefly, in vivo spermatocytes were yielded from seminiferous tubules as previously described (31 (link)). Then these spermatocytes were washed three times with 1x phosphate buffered saline (PBS) and diluted in 200 μL PBS/1% BSA containing 30,000 to 50,000 cells for each Cytospin spot and spun for 7 minutes at 112g. Similarly, in vitro-induced germ cells were detached from the culture dish using 0.25% trypsin, and transferred to microscope slides by Cytospin. The slides were air dried for 10min, fixed in 4% PFA and stored at 4°C in PBS or stored at -80°C after air drying.
Immunocytochemistry was performed as described previously (17 (link)). Omission of the primary antibodies and replacement with mouse, rabbit and sheep isotype IgGs were used as negative control. Primary antibodies and secondary antibodies are described in Supplementary Table 2.