Embryonic stage was determined as E11.5 on the basis of the number of somites, while the sex of the embryos was determined by polymerase chain reaction (PCR) for the Sry gene using the following primer pairs: Sry-F: 5′-ggttgcaatcataattcttcc-3′ and Sry-R: 5′-cactcctctgtgacactttag-3′.
For section immunostaining, embryonic gonads were fixed with 4% paraformaldehyde overnight at 4°C and then embedded in paraffin. Whole gonads were sectioned (6 μm) and autoclaved with Antigen Unmasking Solution (Vector Laboratories, Inc., USA). After the samples were subjected to blocking with 5% skim milk in PBS, they were incubated overnight at 4°C with primary antibodies against deleted in azoospermia-like (DAZL; 1:1,800) [14 (link)] and NANOG (1:5,000; IHC-00205, Bethyl Laboratories, Inc., USA) in Can Get Signal immunostain (NKB-501; Toyobo Co., Ltd., Japan). After the samples were washed, they were incubated with Alexa 488- or Alexa 594-conjugated IgG antibodies at 25°C. The sections were enclosed in Gel/Mount (Biomeda Corp., USA) and observed using fluorescence microscopy (Axio Imager M2, Carl Zeiss, Germany). The number of germ cells in each genital ridge section was assessed by Image J (version 1.50i).
For section immunostaining, embryonic gonads were fixed with 4% paraformaldehyde overnight at 4°C and then embedded in paraffin. Whole gonads were sectioned (6 μm) and autoclaved with Antigen Unmasking Solution (Vector Laboratories, Inc., USA). After the samples were subjected to blocking with 5% skim milk in PBS, they were incubated overnight at 4°C with primary antibodies against deleted in azoospermia-like (DAZL; 1:1,800) [14 (link)] and NANOG (1:5,000; IHC-00205, Bethyl Laboratories, Inc., USA) in Can Get Signal immunostain (NKB-501; Toyobo Co., Ltd., Japan). After the samples were washed, they were incubated with Alexa 488- or Alexa 594-conjugated IgG antibodies at 25°C. The sections were enclosed in Gel/Mount (Biomeda Corp., USA) and observed using fluorescence microscopy (Axio Imager M2, Carl Zeiss, Germany). The number of germ cells in each genital ridge section was assessed by Image J (version 1.50i).