Eosin solution

Heart tissues were harvested and sliced across the papillary muscles, then fixed with 10% formalin and embedded in paraffin. Five-μm-thick sections were de-paraffinized and rehydrated. After rehydration in 70% ethanol, the sections were incubated in filtered Mayer’s hematoxylin solution (Cat# 131-09665, Wako, Osaka, Japan) and washed with tap water. Then the sections were incubated in eosin solution (Cat# 051-06515, Wako), dehydrated with an ethanol series (70%, 80%, 90%, and 100%), cleared in xylene, and mounted in MGK-S mounting medium (Cat# FK00500, Matsunami, Osaka, Japan). For picrosirius red staining, the sections were incubated with freshly prepared staining buffer (1.2% picric acid [Cat #88-89-1, Wako], 0.1% Fast Green FCF [Cat #F7252, Sigma-Aldrich], and 0.1% Direct Red 80 [Cat #365548, Sigma-Aldrich]) for 1 h at room temperature. Sections were washed briefly in distilled H₂O and dehydrated. The slides were mounted in MGK-S mounting medium. Images were taken with a Leica M165C microscope (Wetzlar, Germany) to examine global change in heart size. The images were analyzed with Image J software to quantify fibrosis. Interstitial fibrosis was calculated by removing the perivascular fibrosis area is expressed as a percentage of the total tissue section area^{43 (link)}.

Female pregnant mice were sacrificed on the following days of pregnancy 14.5, and 18.5. Embryos and placentas were collected and weighed. Placentas were fixed in 4% paraformaldehyde (Wako,
Osaka, Japan) in PBS, embedded in paraffin, and sectioned at 8 μm thickness. Specimens were stained with Mayer hematoxylin solution (Wako) and Eosin solution (Wako). Specimens were mounted
in Permount (Falma, Tokyo, Japan) and analyzed with a BZ-X710 microscope (Keyence, Osaka, Japan).

Mouse brains were fixed with 4% paraformaldehyde for 12 h, washed with PBS, and embedded in paraffin. Sagittal or coronal sections (5-μm thickness) were made using a microtome (Yamato Kohki Industrial Co., Ltd.). The sections were de-paraffinized by xylene, rehydrated, washed with water, and stained with Carrazzi’s Hematoxylin solution (1.15938.0025; Merck) for 10 min at room temperature. After washing with tap water for 10 min, sections were stained with eosin solution for 5 min at room temperature (0.25% eosin diluted with 80% ethanol, 051-06515; Wako) and dehydrated with ethanol. Sections were immersed with xylene for clearing and covered. Images were obtained by light microscopy (BX53; Olympus).

Frozen plantaris muscles were cut as 10‐µm sections using a cryostat (ThermoFisher Scientific K.K, Tokyo, Japan). Sections on the slide glasses were soaked in Mayer's hematoxylin solution (FUJIFILM Wako Pure Chemical Co.) for 10 min, washed with warm water, and then incubated with eosin solution (FUJIFILM Wako Pure Chemical Co.) for 30 s. Sections were dehydrated in 100% ethanol and mounted using Fluorescent Mounting Media (SeraCare Life Science Inc, Milford, MA, USA). Muscle fiber cross‐sectional area (CSA) was measured by circling each fiber per muscle using the Fiji image processing package based on the ImageJ Software (Schindelin et al., 2012).

The sample stained with the hematoxylin solution (FUJIFILM Wako Chemicals Corporation, Osaka, Japan) at 22 ± 2 °C for 5 min, washed with running tap water for 10 min, and then stained with eosin solution (FUJIFILM Wako Chemicals Corporation) at 22 ± 2 °C for 3 min.