Paraformaldehyde (pfa)

At 72 h after modeling, distal ileum specimens (~1 cm) were collected, fixed in 4% paraformaldehyde (Wuhan Boster Biological Technology, Ltd.) for 24 h at room temperature and embedded in paraffin. Then, 5-µm thick sections were cut and stained with H&E. The sections were stained with hematoxylin for 5 min and then dehydrated with 70 and 90% ethanol, followed by staining with eosin for 3 min; Images were obtained using a microscope (Olympus BX53; Olympus Corporation). Histopathology was assessed according to the Chiu grading system, and histopathological scores (0-5 scale) were calculated following observation by light microscopy at ×200 magnification. The Chiu scoring standard of the ileal mucosa is divided into six levels according to the severity of injury: i) 0, the intestinal mucosal epithelium is normal without injury; ii) 1, the intestinal mucosal subepithelial space is widened and the capillaries have hyperemia; iii) 2, the intestinal mucosal subepithelial space is further extended and elevated, and separation of the epithelium and lamina propria occurs; iv) 3, part of the villous epithelium of the intestinal mucosa falls off; v) 4, the intestinal mucosal villous epithelium completely falls off, and capillary hemorrhages can be observed; and vi) 5, the intestinal mucosal lamina propria disintegrates, and mucosal bleeding ulcer occurs (21 (link),22 ).

The cartilage tissues from rats in the sham or OA groups were fixed with 4% paraformaldehyde (Wuhan Boster Biological Technology, Ltd.) at room temperature for 24 h, embedded in paraffin, cut into 4-µm thick sections and visualized under light microscope (Leica Microsystems GmbH) at x200 magnification after H&E staining.

Samples of 80 tumor tissues and 80 paracancerous epithelial tissues were selected for the construction of the tissue microarray. All samples were fixed with 4% paraformaldehyde (Wuhan Boster Biological Technology, Ltd., Wuhan, China), embedded in paraffin (Wuhan Boster Biological Technology, Ltd.), cut to the desired thickness of 3 µm, and affixed to slides. The slides were stained with hematoxylin and eosin (Wuhan Boster Biological Technology, Ltd.) and assessed by two histopathologists. Two representative tissue cores of each tissue block were selected for transfer to a master block using a manual tissue microarray instrument (ATA-27; Beecher Instruments, Inc., Sun Prarie, WI, USA). The master block was cut to the desired thickness of 4 µm, and sections were placed on 3-aminopropyltriethoxysilane-coated slides (Wuhan Boster Biological Technology, Ltd.).

At 1 week post-fluorescent tracer injection, the ipsilateral DRG of L4, L5 and L6 were removed and fixed in 4% paraformaldehyde (Wuhan Boster Biological Technology, Ltd.). The DRGs were then dehydrated, embedded in optimal cutting temperature compound (Applygen Technologies Inc., Beijing, China) and sectioned coronally using a microtome (Leica CM1510S; Leica Microsystems, Wetzlar, Germany). The fluorescence was examined under a fluorescent microscope (Olympus IX71; Olympus Corporation, Tokyo Japan), and the numbers of FR- and FG-positive neurons were analyzed using ImagePro Plus version 6.0 (Media Cybernetics, Inc., Rockville, MD, USA).

Migration assays were performed in Transwell inserts (8-µm pores; Corning, Inc.). Briefly, 700 µl fTEB-CM was added to the lower chamber. A total of 2x10⁴ hBMSCs in 200 µl serum-free medium (Cytiva) were seeded in the upper chamber and allowed to migrate for 48 h at 37˚C. Next, cells in the upper chamber (non-migrating cells) were removed using a cotton wool swab. Migrated cells in the lower chamber were washed with PBS, fixed with 4% paraformaldehyde (Wuhan Boster Biological Technology, Ltd.), stained with DAPI for 15 min at room temperature and observed under a fluorescent microscope (Olympus Corporation). The number of migrated cells was counted from 10 random high-power fields (magnification x100) and the mean taken. TEB-CM, DBM-CM and DMEM/F12 containing recombinant human SDF-1 (20 ng/ml) served as controls. Migration assays were performed on five batches of hBMSCs from different donors.

The MCF7, T47D, MCF7R or T47DR cells were seeded in the upper chamber of a Transwell insert (3×10⁵ cells/well; EMD Millipore) coated with Matrigel (1:7.5) in 200 µl of serum-free medium. Supplemented RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) was added to the lower chamber. The cell lines were incubated for 24-48 h at 37°C, and the cells were fixed with 4% paraformaldehyde (Wuhan Boster Biological Technology, Ltd.) for 15 min at room temperature and stained with 0.5% crystal violet (Wuhan Boster Biological Technology, Ltd.) for 5 min at room temperature. the number of migrating cells was determined in 5 random fields at an inverted light microscope (magnification, ×200; TE2000-U; Nikon Corp.).

Immunofluorescence was performed according to previously reported methods (10 (link)). Transfected cMSCs (1.5×104 cells/cm2) were fixed with 4% paraformaldehyde (Wuhan Boster Biological Technology, Ltd., Wuhan, China) for 15 min at room temperature, washed in phosphate-buffered saline (PBS; AR0030; Wuhan Boster Biological Technology, Ltd.), then treated with 0.2% Triton X-100 (T8787; Sigma-Aldrich) for 15 min. Cells were incubated with rabbit polyclonal anti-HCN4 antibody (1:100; ab69054; Abcam, Cambridge, UK) overnight at 4°C. Following washing three times with PBS for 5 min, the cells were incubated with Alexa FluorTM 647-conjugated donkey anti-rabbit IgG (1:200; A-31573; Invitrogen) for 60 min at 25±1°C. After further washing with PBS, the cells were mounted with Antifade Mounting Medium (Beyotime Institute of Biotechnology, Shanghai, China). Nuclei were stained with 4′,6-diamidino-2-phenylindole (D9542; Sigma-Aldrich) as a location control. Fluorescent images were obtained using an inverted laser confocal microscope (LSM 710; Carl Zeiss Microscopy GmbH, Cologne, Germany). The results were analyzed using ZEN lite software, 2011 edition (Carl Zeiss Microscopy GmbH).