Microtome

Testicular tissue was decapsulated of tunica albuginea, washed with PBS, and xed in 4% paraformaldehyde. Next, 8-10 µm thick sections were prepared using a microtome (BRAND, COUNTRY). The sections were mounted on Hydrophilic Plus slides and stored at room temperature until used. For the immunohistochemical staining process, slides were washed by xylene before being placed in a series of decreasing concentrations of ethanol in water. Before staining, antigen retrieval was done by heatinduced epitope retrieval (HIER) methods at 95°C for 20 minutes, and the non-speci c binding sites in the tissue sections were blocked with 10% serum/0.3% Triton in PBS. The prepared slides were divided into two groups and incubated with either primary sox9 antibody or vimentin antibody (Sigma) and species-speci c secondary antibodies. The characterization of labelled cells was analyzed by confocal laser scanning microscope Zeiss LSM (Zeiss, Germany) [20] .

Testicular tissue was decapsulated of tunica albuginea, washed with PBS, and xed in 4% paraformaldehyde. Next, 8-10 µm thick sections were prepared using a microtome (BRAND, COUNTRY). The sections were mounted on Hydrophilic Plus slides and stored at room temperature until used. For the immunohistochemical staining process, slides were washed by xylene before being placed in a series of decreasing concentrations of ethanol in water. Before staining, antigen retrieval was done by heatinduced epitope retrieval (HIER) methods at 95°C for 20 minutes, and the non-speci c binding sites in the tissue sections were blocked with 10% serum/0.3% Triton in PBS. The prepared slides were divided into two groups and incubated with either primary sox9 antibody or vimentin antibody (Sigma) and species-speci c secondary antibodies. The characterization of labelled cells was analyzed by confocal laser scanning microscope Zeiss LSM (Zeiss, Germany) [20] .