Eosin y solution

Maxillae from the mice were fixed in 4% paraformaldehyde (Wako Pure Chemical Industries Ltd., Osaka, Japan) overnight at 4°C and decalcified in decalcify solution B (Wako Pure Chemical Industries Ltd.) for 1 week. After decalcification, periodontal tissues were embedded in paraffin and sectioned at 5 μm in a mesio-distal orientation using a LEICA RA2245 microtome (Leica Microsystems, Wetzlar, Germany). Hematoxylin—eosin staining was performed using Mayer’s hematoxylin solution (Muto Pure Chemicals Co., Ltd., Tokyo, Japan) and 1% Eosin Y solution (Wako Pure Chemical Industries Ltd.).

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.

Cryosections were washed three times with PBS and soaked in Mayer's hematoxylin solution (Wako) for 5 min. The slides were washed with water for 5 min, transferred into eosin-Y solution (Wako) for 20 sec, washed twice with water and mounted in the mounting medium. Images were obtained by using LSM710.

An anti-CD68 antibody, 1× Tris-buffered saline with Tween® 20, SignalStain® antibody diluent, SignalStain® Boost IHC detection reagents, and SignalStain® DAB substrate kits were purchased from Cell Signaling Technology (Danvers, MA, USA). Xylene, mounting medium, Mayer’s hematoxylin solution, and 1% Eosin Y solution were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Goat serum was purchased from Merck (Darmstadt, Germany).

Paraffin-embedded samples were sectioned at 5 µm. The sectioned samples were deparaffinized, rehydrated, and stained with Mayer's hematoxylin solution (Wako Pure Chemical Industries, Ltd., Osaka, Japan) and 1% Eosin Y solution (Wako Pure Chemical Industries, Ltd.). After washing, the samples were mounted with Softmount (Wako Pure Chemical Industries, Ltd.).

The mouse placenta was fixed with 4% PFA in water overnight at 4°C and then embedded in paraffin. Paraffin sections (7 μm thick) of the central (maximum diameter) region of the mouse placenta were deparaffinized and stained with Mayer's Hematoxylin Solution (#131-09665; FUJIFILM Wako Pure Chemical Corporation) and 1% Eosin Y Solution (#051-06515; FUJIFILM Wako Pure Chemical Corporation) according to the manufacturer's instructions. Cover glasses were mounted with Permount Mounting Medium (#SP15-100; Fisher Scientific, Pittsburgh, PA, USA) after dehydration. Images were collected using an all-in-one fluorescence microscope (BZ-X700; KEYENCE Corporation, Osaka, Japan).

Paraffin-embedded samples were sectioned at 5 µm. The sectioned samples were deparaffinized, rehydrated, and stained with Mayer’s hematoxylin solution (131-09665; Wako Pure Chemical Industries) at RT for 5 min and 1% eosin Y solution (051-06515; Wako Pure Chemical Industries) at RT for 2 min. After washing, the samples were mounted with Softmount (192-16301; Wako Pure Chemical Industries). Images of sample were visualized using BZ-810 (Keyence, Osaka, Japan) and analyzed with BZ-800 analyzer software (Keyence).