G1012

Three ventricular sections from each rat were incubated with individual primary antibodies to CD68 (GB11067; 1/3,000; Servicebio Technology, Wuhan, China), CD163 (ab182422; 1/500; Abcam), or CD86 (13395‐1‐AP; 1/500; Wuhan Sanying). Subsequently, sections were incubated with secondary antibodies conjugated with fluorescence. The slides were washed three times with PBS, incubated in 4′,6‐diamidino‐2‐phenylindole (G1012; 1:1; Servicebio Technology) for 10 min, and then dried and coverslipped for evaluation. Fluorescent signals of 5 random nonoverlapping fields were captured with a fluorescence microscope (Nikon Eclipse C1; Nikon, Tokyo, Japan). All images were analyzed by Image‐Pro Plus 6.0 software. Quantification was performed by calculating the percentage of the positively stained area to the total area at a ×400 magnification.

Ectopic endometrium, eutopic endometrium and normal endometrium were fixed, embedded and sliced. After deparaffinizing and rehydrating the paraffin sections [39 (link),40 (link)], they were placed in a repair box filled with citric acid antigen retrieval buffer (pH 6.0) for antigen retrieval. Next, sections were placed in 3% hydrogen peroxide and incubated at room temperature for 25 min to block endogenous peroxidase activity, followed by serum blocking with 3% BSA (Servicebio G5001, Wuhan, China) for 30 min at room temperature. Anti-human IgM rabbit monoclonal antibody (1:1000 dilution; HUABIO, Cambridge, MA) was incubated overnight at 4 °C, followed by an incubation with secondary antibody at room temperature for 50 min. After the addition of secondary antibody, the sections were incubated with DAPI (Servicebio G1012, Wuhan, China) solution for 10 min at room temperature, and then spontaneous fluorescence quenching reagent was added and incubated for 5 min. Then, cover slips were mounted with anti-fade mounting medium, and images were captured by fluorescence microscopy.

The muscle tissue was removed from 4% paraformaldehyde and dehydrated with sucrose solution. After frozen section, the sections were sealed with 3% BSA for 30 min, and then incubated with the first antibody (Servicebio, GB 112130/GB 111875, 1:3000/1:500) at 4 °C overnight and the fluorescent second antibody at room temperature for 50 min (Servicebio, gb21303/gb25301, 1:300/1:400). The nuclei were dyed with DAPI (Servicebio, g1012). The images were taken under an inverted fluorescence microscope (Olympus Bx-51, Tokyo, Japan) at ×200 magnification. Eight fields were randomly selected in each group, and the cross-sectional area of muscle fibers was calculated by Caseviewer 2.1. The average cross-sectional muscle fiber area was calculated as the total cross-sectional area/total number of muscle fibers in the visual field.

HMC-1 cells were fixed with 4% paraformaldehyde for half an hour. After washing with PBS 3 times, the cells were blocked with 5% BSA for 1 h. In a 4 °C refrigerator, the cells were incubated with PD-1 antibody (1:100, 18106-1-AP, Proteintech) for 1 h. After washing with TBST 3 times, the cells were incubated with cy3-conjugated goat anti-rabbit IgG (1:500, GB11013-1, Servicebio) for 1 h. Then, the cells were washed with TBST 3 times, and the nuclei were dyed with DAPI (G1012, Servicebio) for 10 min. After washing with TBST 3 times, the cells were resuscitated with anti-fluorescence quenching agent (G1401, Servicebio), the cell suspension was dropped onto a glass slide, and then pictures were taken with a laser confocal microscope. For sections of paraffin-embedded mouse tumors, we used chymase antibody (1:800, GB111085, Servicebio) to show mast cells and PD-1 antibody (1:2000, GB11338-1, Servicebio) to show the PD-1 receptor. We confirmed whether BMMCs were induced successfully by labeling with c-kit (1:800, GB11073-2, Servicebio).

The immunofluorescence test was carried out according to a protocol described previously (Wang et al., 2021 (link)). The sperm were washed three times with PBS, smeared onto glass slides and air-dried. The sperm slides were next fixed with 4% formaldehyde in PBS at room temperature for 20 min, followed by washing in PBS three times. Slides were then blocked in 5% bovine serum albumin (A8020; Solarbio, China) and incubated overnight at 4°C with primary antibody anti-CFAP251 (H00144406-B01P, Abnova, China) and HRP-conjugated secondary antibody (GB23301, Servicebio, China). Furthermore, incubation was carried out at room temperature for 10 min with Cy3-tyramide (G1223, Servicebio, China), followed by washing with PBS three times. Then, the slides were incubated with anti-α-tubulin antibodies (GB12200, Servicebio, China) and HRP-conjugated secondary antibody followed by FITC-tyramide (G1222, Servicebio, China). The slides were counterstained with 5 mg/ml DAPI (G1012, Servicebio, China) and mounted with mounting media (G1401, Servicebio, China). Finally, fluorescence images were taken using a confocal microscope (Nikon Eclipse CI, Nikon, Japan).

Paraffin sections were dewaxed in xylene and rehydrated in ethanol and distilled water. After antigen retrieval with EDTA, sections were blocked in 3% BSA for 30 min at room temperature. The blocked solution was gently shaken off and the primary antibody, namely, Anti -CDKN2A/p16INK4a Rabbit pAb) (GB111143, Servicebio, Wuhan, China) was added to the sections and incubated overnight at 4°C. The next day, a secondary antibody (Cy3 conjugated Goat Anti-Rabbit IgG, GB21303, Servicebio, Wuhan, China) was added and incubated at room temperature for 1 h. After DAPI (G1012, Servicebio, Wuhan, China) stained the nuclei, the sections were dried slightly and then sealed with anti-fluorescence quenched sealing tablets. Images of the cortex were captured at ×20 magnification with a fluorescence microscope (Nikon DS-U3, Nikon).

Different treatments for the U251 cells were applied according to the manufacturer’s protocols after 24 h. First, the intracellular Ca²⁺ with Fluo-4 AM was fluorescently stained and then incubated with MitoSOX (5 μM) at 37 °C for 10 min in the dark, washed twice with Hanks with calcium magnesium (1 × HBSS, PH1509, Phygene) pH 7.4, and pre-cooled at 4 °C in 4% paraformaldehyde and fixed for 10 min at RT. Other U251 cells were stained with Fluo-4 AM and then blocked with 10% normal donkey serum (G1217, Servicebio, Wuhan, China) for 30 min at RT and were then incubated with rabbit anti-Piezo1 antibody (1:200, 15939-1-AP, Proteintech, WuHan, China) at 4 °C overnight (about 16 h). The next day, the fluorescence-labeled secondary antibodies were added (1:400, 711-545-152, Alexa Fluor 488-ChromPure Donkey IgG, Jackson ImmunoResearch, West Grove, PA, USA; according to the instructions, 0.5 mg was rehydrated with 0.4 mL ddH₂O at an antibody concentration 1.5 mg/mL) for 1 h at RT in the dark. Finally, they were washed with PBS three times (5 min each time) and were then incubated with DAPI (G1012, Servicebio) at RT in the dark for 15 min and then washed three times again. The cell nucleus was stained with Hoechst 33342 (CSA: C1028, Beyotime) for 10 min at 37 °C in the dark, and images were captured with a confocal microscope (ZEISS LSM 880, Oberkochen, Germany) (×40 oil immersion lens).