Mayer s hematoxylin solution

The plantaris muscle was covered in optimal cutting temperature (OCT) compound (Sakura Finetek, Tokyo, Japan), and then quickly frozen in liquid nitrogen-cooled isopentane and stored at −20 °C until sectioning. Frozen muscles were sectioned at thickness 7 μm, air dried, and stored at −20 °C. Images were captured with the Olympus BX-51 microscope (Tokyo, Japan).
To determine the CSA of muscle fibers, the muscle sections were incubated with Mayer’s hematoxylin solution (Wako, Osaka, Japan) for five min to stain the nuclei and then washed with water for one min. Following this, they were stained with eosin solution (Wako, Osaka, Japan) for one min and then washed with water for one min. The stained sections were observed under the BX-51 microscope and CSA analysis was carried out using the Image J software.

Testes were fixed in 4% paraformaldehyde in PBS and embedded in Technovit 8100 resin (Heraeus-Kulzer, Wehrheim, Germany) according to the manufacturer’s instructions. Sections were cut at 5 μm and stained with periodic acid-Schiff (PAS) and then counterstained with Mayers hematoxylin solution (Wako, Japan).

Musculus longissimus lumborum was paraffin-embedded and sectioned at a 5-μm thickness, as described previously [17 (link)]. Paraffin sections were deparaffinized with a lemosol solution (Fujifilm Wako Chemicals, Osaka, Japan). Collagen fiber staining was performed using a Picro-Sirius Red stain kit (ScyTek Laboratories, Logan, UT, USA).
For tissue immunostaining, deparaffinized sections were activated with an antigen-retrieval solution (Histo VT one solution; Nacalai Tesque, Kyoto, Japan) at 90 °C for 20 min. After permeabilization with 0.3% Triton-X in PBS (−) for 30 min and inactivation of endogenous peroxidase with 0.3% H₂O₂ solution for 30 min, the sections were treated overnight with an antibody diluted with Can Get Signal immunostaining solution A (Toyobo). The bound antibody was visualized using Histofine Max-Po (Nichirei Biosciences, Tokyo, Japan) and ImmPACT DAB (Vector Laboratories, Burlingame, CA, USA). Counterstaining was performed with Mayer’s hematoxylin solution (Fujifilm Wako Chemicals), and image analysis was performed using an all-in-one microscope system (BZ8000; Keyence, Osaka, Japan).

After the fertility test, three knockout male mice and three same-aged wild-type B6D2F1/J mice were euthanized by cervical dislocation following anesthesia to examine their testis weights, testicular and epididymal histology, sperm morphology, and sperm motility. Testes and epididymides were fixed in Bouin fluid (Polysciences, Inc., Warrington, PA), embedded in paraffin wax, sectioned 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's reagent (Wako, Osaka, Japan) followed by counterstaining with Mayer's hematoxylin solution (Wako, Osaka, Japan). Spermatozoa were extracted from cauda epididymis and dispersed in TYH medium for 10 min. Sperm morphology was observed under an Olympus BX53 differential interference contrast microscope equipped with an Olympus DP74 color camera (Olympus, Tokyo, Japan). Sperm motility was measured with the CEROS II sperm analysis system (Hamilton Thorne Biosciences, Beverly, MA) at 10 min and 2 h of incubation.

Samples embedded in paraffin were sliced at 5 μm and the sliced samples were then deparaffinized, rehydrated, stained with Mayer’s hematoxylin solution (Wako Pure Chemical Industries, Osaka, Japan) and 1% Eosin Y solution (Wako Pure Chemical Industries) and mounted with Softmount (Wako Pure Chemical Industries).

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).