*2.8. Immunohistochemistry (IHC)*/*Immunocytochemistry (ICC)*

For IHC, PND3 testis samples were fixed with 4% paraformaldehyde solution (PFA), paraffin embedded, and 4 to 6 μm thick sections were made. IHC and ICC analyses were performed as previously described [18,19,23,25]. Briefly, the slides were dewaxed, rehydrated, treated for antigen retrieval, and then with a blocking reagen<sup>t</sup> in PBS. The slides were then incubated overnight at 4 ◦C in Anti PRDX6 primary antibody diluted (1:100) in PBS containing 0.1% Triton X-100 and serum. This was followed by washes and 1-h incubation with a biotinylated secondary antibody (dilution 1:100) in 0.1% Triton X-100 and 10% BSA, at room temperature, then 15 min of treatment with Streptavidin-horse radish peroxidase and 15 min with an AEC Chromogen solution. Mayer's hematoxylin was used for counterstaining, and Clear-Mount for coating. The slides were examined using bright-field

microscopy. As negative controls, some slides were treated with non-specific immunoglobulin G instead of specific antibody.

For ICC, the protein expression of PRDX1, 2, and 6 were examined in low purity gonocyte fractions pooled, washed with PBS, and fixed with 3.5% paraformaldehyde right after the BSA gradient. The fixed cells were collected by cytospin centrifugation, the slides dried and treated with acetone:methanol (60:40), followed by the antigen retrieval solution. The ICC reactions were similar to those described above for IHC, except for the use of fluorescent secondary antibodies. DAPI was used as a nuclear signal. Rabbit and mouse IgG were used as negative controls and gave no fluorescent signal (data not shown). Pictures of fluorescent signals and bright field were taken, using the same time of exposure for the specific antibodies and non-specific IgGs. Representative samples are shown.
