Paraformaldehyde (pfa)

Myocytes apoptosis in cardiac tissue was examined by terminal dUTP trasferase nick end labeling (TUNEL). The paraffin- embedded 5-μm thick sections of cardiac tissue were digested by proteinase K (20 μmol/L, Sigma) and fixed by paraformaldehyde (4 %, Solarbio) and then permeabilized by Triton X-100 (0.1 %, Sigma- Aldrich). A TUNEL assay kit (Roche) was employed to detect the apoptotic cells per manufacturer’s instructions. Briefly, the cryo- sections were washed by PBS and then incubated with prepared TUNEL solution for 1 h at 37 °C in a dark chamber. The TUNEL-positive cells were observed and counted under a light microscope (Motic). For in vitro apoptosis evaluation, the In Situ Cell Death Detection kit (Roche) was used. Briefly, the cultured myocytes were fixed with paraformaldehyde (4 %, Solarbio) and then permeabilized with Triton X-100 (0.1 %, Sigma- Aldrich). According to the manufacturer’s protocols, the cells were incubated with TUENL staining solution for 1 h. The nuclei were stained with DAPI (Beyotime). The TUNEL positive cells were observed and counted under a fluorescence microscope (Nikon).

Mature adipocytes were stained with Oil Red O (ORO) to confirm differentiation efficiency. Cells were fixed with 4% paraformaldehyde (PFA) (Solarbio, Beijing, China) for 30 min at room temperature or overnight at 4°C. Next, the working solution of ORO (Sigma‐Aldrich, St. Louis, MO, USA) (Oil Red O: deionized water = 4:6) was prepared and kept away from light at room temperature. Then, 60% isopropanol was used to wash the cells. The cells were stained with the working solution for 15–20 min at room temperature. After staining, the cells were washed twice with distilled water. Finally, the cells were examined microscopically and photographed (Nikon, Tokyo, Japan).

The cells were fixed with 4% paraformaldehyde (PFA, Solarbio, China) for 15 min, washed with PBS, permeabilized with 0.5% Triton X-100 for 30 min, and blocked with 5% goat serum (Solarbio, China) for 1 h at room temperature. Then, the cells were incubated with the primary antibodies in blocking buffer overnight at 4°C. After washing three times with PBS, the cells were incubated with the fluorescence-labeled secondary antibodies Alexa Fluor® 555 (1:500, ab150078, abcam), Alexa Fluor® 488 (1:500, ab150113, abcam) or CoraLite® 488 (1:500, SA00013-2, Proteintech) for 2 h at room temperature in the dark. A staining solution consisting of 4',6-diamidino-2-phenylindole (DAPI, Solarbio, China) was added to stain the nuclei, and the images were collected under a fluorescence microscope (Olympus, Japan). For cell immunofluorescence staining counts, the number of samples per group was n=5, and 5 fields of view were randomly captured for each sample. The primary antibodies used in this study were as follows: Nestin (1:200, PA5-118114, Invitrogen), GAP-43 (1:500, ab16053, abcam), Sox-2 (1:300, 11064-1-AP, Proteintech), GFAP (1:500, ab10062, abcam), βIII-tubulin (1:500, 66375-1-Ig, Proteintech), Iba-1 (1:500, 019-19741, Wako), NLRP3 (1:200, YT5382, Immunoway), and Caspase-1 (1:300, YT5743, Immunoway).

Fourteen women aged 23-41 years (31.3 ± 6.5 years) donated small ovary cortical biopsy specimens (adjacent nonpathological tissue) while undergoing routine gynecological laparoscopies at Guangzhou First People's Hospital, Guangzhou, Guangdong, China. We obtained informed consent before surgery, and the study was conducted in accordance with the Declaration of Helsinki. The collection and use of human ovarian tissue were approved by the ethics committee of Guangzhou First People's Hospital (No. K-2021-184-01). The ovary tissues were immediately transported to the laboratory within cold preequilibrated Dulbecco's phosphate-buffered saline with ITS and penicillin-streptomycin.
The ovary tissues were divided into fragments of ~1 mm³ in sterile conditions. Some fragments from each tissue were collected for protein detection or were fixed in 4% paraformaldehyde (PFA, Solarbio, Beijing, China) for serial sections and then stained with hematoxylin for follicle counting (uncultured group). The other fragments were divided equally and randomly for culture in medium (control) supplemented with 0.05 μM TG, 5 μM CrCl₃, 200 μM MnCl₂, 15 μM FeCl₃, or 35 μM ZnSO₄ for 4 days and then in drug-free medium for another 2 days. The fragments were collected to analyze the protein levels after 4 days of culture or to count the follicle number after 6 days of culture.

The tumor samples were fixed with 4% (PFA; Solarbio), and embedded in paraffin. Multiple sections (5.0-µm thick) were cut from the paraffin-embedded NSCLC tissues. After that, the samples were incubated with primary antibody (MTDH, ab45338) overnight at 4°C. Next, the corresponding secondary antibody was selected to amplify the signals via incubation for 30 min at room temperature.

To evaluate the effect of the formation of clot network and the release of blood components on initial cell adhesion and proliferation, the BMSCs were seeded at an initial density of 2 × 10⁴ cells/mL on the control or pre-incubated titanium slices which were placed in the bottom of 24-well plates and cultured with Dulbecco’s modified eagle medium (DMEM, Hyclone) + 10% FBS. After 4 and 24 h culture, adherent cells were fixed with 4% paraformaldehyde (PFA, Solarbio) and stained with rhodamine-phalloidin (Cytoskeleton, Inc.) and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI). The cell morphology was observed and imaged by a laser confocal scanning microscope (LCSM, Nikon A1, Japan).
The proliferation of cells was detected by AlamarBlue assay (Invitrogen, Grand Island, NY) at 1, 3, and 7 days. Briefly, the cells were incubated on control or pre-incubated surfaces for 3 h in a medium complemented with 10% (v/v) AlamarBlue reagent. The optical density was valued (λex = 540 nm, λem = 590 nm) by a SpectraMax M5 (Molecular Devices, China). The results were analyzed by plotting optical density against cell concentration.

hPDLCs seeded on chamber slides were subjected to force and/or LIPUS, followed by osteogenic induction for 7 days. The cells from different groups underwent fixation at 4°C for 15 min using 4% polyformaldehyde (PFA, Solarbio), followed by permeabilization for 5 min using 0.1% Triton X-100 (Solarbio). After a PBS wash, the cells were subjected to blocking with 5% bovine serum albumin (BSA, Solarbio) at room temperature for 60 min. Subsequently, the cells were subjected to immunostaining, employing a primary antibody, anti-Piezo1 (1:200, 15939-1-AP; Proteintech), overnight at 4°C. Following this, the cells were incubated with a Cy3 fluorescent secondary antibody (1:200, SA00009-2, Proteintech) at room temperature for 1 h. Subsequently, after PBS washing, the cells were sealed using a mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI, Solarbio). Images were captured using an inverted confocal microscope (FV3000, Olympus, Tokyo, Japan).