Histology of mouse tissue sections

Adult males (post-natal day 60, P60) were euthanized using CO2 and testes were isolated. Pictures of freshly isolated testes were taken with SteREO Discovery V12 (Zeiss) (Figure 6C). To prepare the paraffin sections, isolated testes were fixed in Bouin’s solution (Sigma, cat. no. HT10132) for 48h at 4°C. Next, testes were washed in PBS for 48 h at 4°C, with frequent PBS changes. Samples were transferred into the embedding cassettes (Simport; cat. no. M508-3) and sent to the histology platform of University of Geneva. The samples were dehydrated in 70% (2 × 2h), 90% (1h), 95% (1h) and 100% ethanol (3 × 30 min) followed by incubation (3 times for 30 min) in HistoSAV2 (Biosystems). The solution was removed, replaced with paraffin, and incubated at 56-58°C. Testes were then transferred into plastic molds (Peel-A-Way® Embedding Mold S22, Polysciences, ; cat. no. 18646A-1) filled with paraffin and left at room temperature for paraffin to solidify. The sections (5 μm thickness) were cut using microtome (Leica RM2135) and mounted on the Superfrost Plus microscope slides (Thermo Fisher; cat no. 4951PLUS4). The sections were allowed to stretch for 24 h at 42°C and then were stored at room temperature.

Next, sections were stained using Hematoxylin and Eosin (H&E) stain protocol. The slides containing the paraffin sections were placed in a glass slide holder filled with HistoSAV2 (3 × 5 min) to remove the paraffin. For rehydration, the slides were incubated in 3x 100% ethanol, 96% ethanol, 70% ethanol, 50% ethanol and water (3 min for each step). Sections were stained with Hematoxylin solution (Merck) for 3-5 min and rinsed in running tap water. Then, sections were stained with Eosin Y solution (Sigma Aldrich; cat. no. E4382) for 3 to 5 min and washed with water. For dehydration, the sections were incubated in 50% (30 s), 70% (30 s), 96% (30 s), 100% ethanol (2 min) and HistoSAV (3 × 3 min). Neo-Mount (Merck) was put on the sections and immediately covered with coverslips. Pictures were taken using microscope AXIO Imager M2 (Zeiss).