In situ cell death pod kit

For apoptosis quantification, ectopic tissue sections were processed for terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-fluorescein nick-end labeling (TUNEL) staining using the “In Situ Cell Death POD” kit (Roche, Basel, Switzerland). Sections were treated according to the manufacturer's instructions. Briefly, sections were deparaffinized in xylene, rehydrated through graded alcohols and permeabilized with 20 μg/ml Proteinase K (Gibco, Grand Island, NY, USA). Endogenous peroxidase was inactivated by coating the samples with 3% H₂O₂. Sections were rinsed with PBS, and then immersed 60 min in TdT buffer at 37°C. Sections were incubated 30 min with the anti-fluorescein peroxidase antibody, followed by the peroxidase substrate DAB. Finally, sections were counterstained with hematoxylin. Slices of female rodent mammary gland obtained 3–5 days after weaning of pups were used as a positive control. As a negative control, a number of tissue samples were subjected to treatment without TdT.
The percentage of TUNEL positive cells was established using a standard light microscope by two independent observers at 400X and any nuclear brown stain was considered as positive. At least 600 epithelial and 600 stromal cells were counted per mouse considering all lesions. Independently of the number of cells at least 10 representative fields were counted per mouse.

TUNEL assays were performed using the In Situ Cell Death Kit POD, following the manufacturer's instructions (Roche, Mannheim, Germany). Then, the sections were postfixed with 4% paraformaldehyde (PFA) for 15 min at RT, incubated in citrate buffer for 20 min and blocked with 5% BSA and 5% horse serum in PBS. The sections were then incubated with Peanut Agglutinin (PNA) lectin conjugated to Alexa Fluor 594 (1:200, L32459, Thermo Fisher Scientific) for 30 min at RT to reveal the acrosome of spermatids and counterstained with Hoechst 33342. Analyses were conducted with an epifluorescence microscope as described above. Spermatid differentiation was evaluated as described by Leblond and Clermont.²² As individual steps were hardly identifiable on cross sections, spermatids were classified into three categories based on the shapes of the nucleus and/or the acrosome: steps 1–7 for round spermatids (round nucleus), steps 8–11 for elongating spermatids (elongating nucleus and acrosome) and steps 12–19 for elongated spermatids (thinner nucleus and hook‐shaped nucleus).

To detect DNA fragmentation and spermatocytes in the same samples, tissue sections were first subjected to TUNEL assays (In Situ Cell Death kit POD, Roche, Mannheim, Germany), following the manufacturer’s instructions. Then, sections were post-fixed with 4% PFA for 15 min at RT and finally underwent the standard immunofluorescence protocol described above (without the permeabilization step). Rabbit anti-SYCP3 antibodies were used, and revealed with secondary antibodies coupled to Alexa Fluor^® 594. Positive controls were performed with DNase I before TUNEL assays and negative controls were carried out by omitting the enzyme solution (TUNEL) or with pre-immune IgGs (SYCP3 immunostaining).