Schiff reagent

Males were sacrificed by cervical dislocation. Testis and epididymis were fixed in 4% paraformaldehyde in PBS and were processed for plastic sectioning using a Technovit 8100 instrument (Kulzer, Wehrheim, Germany) according to the manufacturer’s instruction. Briefly, fixed testes were washed in PBS at 4 °C for 1 h, dehydrated in acetone at 4 °C for 1 h, infiltrated with in mixed solution of Technovit 8100 basic solution and hardener 1 (1.5 mL of basic solution plus 9 mg of hardener 1 per sample) at 4 °C for 2–6 h, and then embedded after adding 50 μl of hardener 2. For analysis of mouse testes, 5 μm sections were treated with 1% periodic acid for 10 min, followed by treatment with Schiff reagent (#193-08445, FUJIFILM Wako Pure Chemical, Osaka, Japan) for 20 min. The sections were stained with Mayer hematoxylin (#131-09665, FUJIFILM Wako Pure Chemical) solution prior to imaging and observed under microscope. For analysis of mouse epididymis, 5 μm sections were treated with Mayer hematoxylin solution for 3 min, then were stained with Eosin solution (1% Eosin Y solution [#051-06515, FUJIFILM Wako Pure Chemical] mixed with 80% ethanol in 1:2 ratio), and then added 0.5% of total volume of acetic acid for 2 min. Stained sections were then observed under microscope.

Formalin-fixed paraffin-embedded tissue sections (5 µm) were first stained with DBA lectin and then subjected to PAS staining. In brief, after antigen retrieval, sections were incubated with biotinylated DBA lectin (1:5000; Sigma) and visualized positive cells with DAB. Next, sections were incubated with 1% periodic acid (Fujifilm Wako Pure Chemical Corporation) for 20 min, with Schiff reagent (Fujifilm Wako Pure Chemical Corporation) for 30 min, and 0.5% sodium bisulfite solution (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) for 5 min. Hematoxylin was used for counter-staining.

Cytospin preparations of X. laevis and X. tropicalis blood cells were stained using May-Grunwald Giemsa solution (MGG; Wako, Osaka, Japan), as previously described^{30 (link)}. O-dianisidine staining was performed as follows: samples were fixed for 5 min in methanol, incubated in 0.1% o-dianisidine and 3% hydrogen peroxide for 1.5 min, washed in running water, and counterstained with Giemsa solution^{31 (link)}. For periodic acid-Schiff (PAS) staining, cells were fixed in 10% formalin-methanol solution for 30 min at room temperature, and washed with tap water. The slides were incubated in 1% sodium periodate for 5 min, and twice washed in distilled water. Glass slides were incubated in the Schiff reagent (Wako, Osaka, Japan) for 15 min at room temperature and then placed in Gill’s haematoxylin solution (Muto Chemical, Tokyo, Japan) for 10 min, washed with tap water, and examined using light microscopy (model BX51; Olympus, Tokyo, Japan).