H8070

The HRGECs were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X (ST795, Beyotime, China). The DNA fragmentation was determined by Terminal deoxynucleotidyl transferase (TdT) -mediated dUTP nick end labeling (TUNEL) via In Situ Cell Death Detection Kit (11,684,955,910, Roche, Swiss) as described by the manufacturer. DAPI is used to locate the nuclei of the cells. Fluorescent images were obtained using the microscope (DP73, OLUMPUS, Japan).
TUNEL staining was performed using a peroxidase In Situ Cell Death Detection Kit (11,684,955,910, Roche, Swiss). Tissue sections were treated with 3% hydrogen peroxide and added the equilibration buffer. Then the sections were treated with TUNEL reaction buffer for 60 min at 37 °C. Specimens were then treated with Converter-POD for 30 min at room temperature and developed with 3,30-diaminobenzidine (DAB) substrate. Sections were counterstained with hematoxylin (H8070, Solarbio, China), rinsed, dehydrated, and mounted. Each slice was observed under a microscope (DP73, OLUMPUS, Japan); the DAB staining intensity was taken to indicate the extent of apoptosis detected by TUNEL.

The distal third of the femur was selected as the region of interest for detection of ALP levels. The samples were embedded in paraffin and cut into 5-μm sections. Then slices were deparaffinated, dehydrated, and stained with ALP staining solution (DE0001, Leagene Biotechnology, China) for 12 h at 37°C. After washing with running water, the sections were counterstained with hematoxylin (H8070, Solarbio, China) for 5 min, and observed under a microscope (BX53, OLYMPUS, Japan).

The paraffin-embedded sections were dewaxed, dehydrated, and washed under tap water for 2 min. After reaction with 3% methanol-H₂O₂ for 20 min, the sections were rinsed with distilled water for 2 min and treated with 0.1 M PBS for 3 min, followed by antigen retrieval. Thereafter, the sections were blocked utilizing normal goat serum (C-0005, Shanghai Haoran Biotechnology Co., Ltd., Shanghai, China) and allowed to maintain at ambient temperature for 20 min. After that, the sections were probed with primary antibodies against STK4 (1 : 150, ab51134, Abcam) and p-YAP1 (ab76252, 1 : 200, Abcam) overnight at 4°C, and then re-probed with biotin-labeled goat anti-rabbit IgG (ab97051, dilution ratio of 1 : 2000, Abcam) at room temperature for 40 min. Subsequently, the samples were stained with 3,3′-diaminobenzidine (DA1010, Solarbio, Beijing, China) for 10 min, followed by 1 min counter-staining by hematoxylin (H8070, Solarbio), dehydration by gradient alcohol, clearing with xylene and mounting with neutral gum. The primary antibody was substituted with PBS as NC. Finally, the sections were observed under an optical microscope (CX41-12C02, Olympus) in 5 randomly selected high-power visual fields. The positive cells were those presenting with brown yellow granules, and the percentage of positive cell was calculated by 2 people blinded to the study.

HE staining was performed to observe the MI‐induced histological changes.23 Two weeks after surgery, the mice from the MI and sham groups were anaesthetized with pentobarbital sodium. Then, heart obtained by thoracotomy was rinsed with phosphate buffered saline (PBS), fixed with 10% formalin for 12 hours and dissected along the ligature to excise myocardial tissues. The paraffin‐embedded tissues were sliced into 4‐μm sections. After being heated at 60°C, the sections were dewaxed with xylene for 20 minutes, dehydrated using gradient ethanol and soaked in distilled water for 5 minutes. Afterwards, the sections were stained with haematoxylin (H8070; Solarbio) for 4 minutes and counterstained with eosin (E8090; Solarbio) for 3 minutes. Finally, the sections were observed under an optical microscope (DSX100, Olympus) and photographed.

Paraffin sections were routinely deparaffinized in xylene and rehydrated in decreased alcohol gradient. After a brief wash with deionized water, tissue antigen was retrieved through heating in sodium citrate buffer for 15 min, followed by incubation with 3% H₂O₂ for 10 min to quench the endogenous peroxidase. Following three times of PBS washing, non-specific antigen-binding sites were blocked with 10% goat serum in PBS (Gibco) overnight. The sections were then incubated with anti-Sox9 (1:5000, EMD Millipore, Cat# AB5535); anti-ki67(1:500, BD, Cat# 550609); anti-MMP-7 (1:100, Cell Signaling Technology, Cat# 3801S); anti-Olfm4 (1:500, Cell Signaling Technology, Cat# 39141S) at 4 °C overnight. After washing in PBS, the sections were incubated with biotinylated goat anti-rabbit IgG (1:1000, Vector Cat# BA-1000) or biotinylated goat anti-mouse IgG (1:1000, Vector Cat# BA-9200) for 2 hr. in room temperature. The detection was performed with a DAB detection kit (Vector Laboratories, Cat# SK-4100) and a VECTASTAIN kit (Vector Laboratories, Cat# PK-7100) according to the manufacturer’s instructions. Following counter-staining in Hematoxylin (Solarbio® LIFE SCIENCE Cat# H8070) and mounting with neutral balsam (Solarbio® LIFE SCIENCE Cat# 96949–21-2). Sections were observed using a light-field microscope (Leica DMI3000B).

Colons from mice were fixed in 4% paraformaldehyde, embedded in paraffin, and then cut into 5 μm thick sections. The sections were stained with hematoxylin (H8070, Solarbio, Beijing, China) for 5 min and eosin (A600190, Sangon, Shanghai, China) for 3 min. Images were observed under a microscope (BX53, Olympus, Tokyo, Japan) at 100x magnification. The histological scores were assessed blindly to evaluate the level of colitis as described by Banerjee et al. [24 (link)].

Antigen retrieval was performed in the lung slices after dewaxing and rehydration, followed by incubation with 3% H₂O₂ (10011218, Sinoreagent, Shanghai, China). The samples were blocked in 1% bovine serum albumin (BSA; A602440-0050, Sangon, Shanghai, China) for 15 min. The sections were incubated with ST2 antibody (1:50; 11920-1-AP, ProteinTech, Wuhan, China) at 4°C overnight and subjected to HRP-labeled goat anti-rabbit IgG (1:500; # 31460, ThermoFisher Scientific, USA) at 37°C for 1 h. Additionally, IL-25R expression was detected in the same way. The sections were incubated with IL-25R antibody (1:100; PA5-47051, ThermoFisher Scientific, USA) at 4°C overnight and next incubated with HRP-labeled donkey anti-goat IgG (1:500; ab6885, Abcam, Cambridge, UK) at 37°C for 1 h. Subsequently, these pieces were stained with 3, 3-diaminobenzidine tetrahydrochloride (DAB; DAB-1031, Maixin, Fuzhou, China) and counterstained with hematoxylin (H8070, Solarbio, Beijing, China). The results were observed under a microscope (Olympus, Tokyo, Japan) at 400 × magnification.