Periodic acid

periodic acid–Schiff (PAS) staining of sections were performed as previously described [30 (link)]. Testes or epididymis were fixed in Bouin solution (Polysciences, Inc., Warrington, PA) and were processed for paraffin embedding. Paraffin sections were cut at a thickness of 5 μm on a Microm HM325 microtome (Microm, Walldorf, Germany) and stained with 1% periodic acid (Nacalai Tesque, Kyoto, Japan) and Schiff reagent (FUJIFILM WakoPure Chemical, Osaka, Japan) followed by counterstaining with Mayer hematoxylin solution (FUJIFILM WakoPure Chemical).

Testes and epididymides were fixed in Bouin solution and embedded in paraffin wax. Paraffin sections were stained with periodic acid (Nacalai Tesque, Kyoto, Japan) and Schiff reagent (Wako, Osaka, Japan) and counterstained with Mayer hematoxylin solution (Wako, Osaka, Japan). The caudal epididymal spermatozoa were dispersed in TYH medium and observed under an Olympus BX53 phase contrast microscopy.

PAS staining of sections were performed as previously described (Morohoshi et al., 2020 (link)). Testes or cauda epididymis were fixed at 4°C in Bouin’s solution (Polysciences, Inc., Warrington, PA, USA) and were processed for paraffin embedding. Paraffin sections were cut at a thickness of 5 μm using an HM325 microtome (Microm, Walldorf, Germany). After rehydrating the sections, they were stained with 1% periodic acid (Nacalai Tesque, Kyoto, Japan) and Schiff’s reagent (FUJIFILM WakoPure Chemical, Osaka, Japan) for 20 min each at room temperature. The sections were then counterstained with Mayer’s hematoxylin solution (FUJIFILM WakoPure Chemical). The sections were observed with an Olympus BX-53 microscope (Tokyo, Japan).

To observe testis gross morphology and measure testicular weight, 11–12 week-old male mice were euthanized after measuring their body weight. The whole testis was observed using BX50 and SZX7 (Olympus, Tokyo, Japan) microscopes. For histological analysis, testes were fixed with Bouin’s fixative (16045–1, Polysciences, Warrington, PA, USA) at 4°C O/N, dehydrated in increasing ethanol concentrations and 100% xylene, embedded in paraffin, and sectioned (5 μm). The paraffin sections were hydrated with Xylene and decreasing ethanol concentrations and treated with 1% periodic acid (26605–32, Nacalai Tesque, Kyoto, Japan) for 10 min, treated with Schiff’s reagent (193–08445, Wako) for 20 min, counterstained with Mayer’s hematoxylin solution (131–09665, Wako) for 3 min, dehydrated in increasing ethanol concentrations, and finally mounted with Permount (SP15-100-1, Ferma, Tokyo, Japan). The sections were observed using a BX53 (Olympus) microscope.

Mouse jejunum sections were fixed in 20% formalin neutral buffer solution, embedded in paraffin, and sectioned into 4-µm-thick sections. After deparaffinization, the sections were treated with an H₂O₂ solution and antigens retrieved by boiling with 10 mM sodium citrate buffer (pH 6.0). After blocking with 5% skimmed milk and 0.005% saponin in phosphate-buffered saline (PBS), the samples were incubated with primary antibodies at 4°C overnight and then with fluorescence or HRP-conjugated secondary antibodies for 30 minutes. For Ulex europaeus agglutinin 1 (UEA-1) staining, UEA-1 (Vector Laboratories, Burlingame, CA, USA) was used instead of the primary antibodies. For ephrinB1 staining, the sections were boiled in 20 mM Tris buffer (pH 9.0) for antigen retrieval and incubated in 1% BSA and 0.005% saponin in PBS for blocking. Chemiluminescence or fluorescence images were recorded on a charge-coupled device camera (Keyence) and a confocal microscope (Leica TCS SPE, Leica Microsystems, Wetzlar, Germany). PAS staining was performed based on standard protocol using periodic acid (Nacalai Tesque, Kyoto, Japan) and Cold Schiff’s reagent (Wako Pure Chemical Industries, Ltd., Osaka, Japan).