Anti fluorescence quenching agent

The second-generation BMSCs were inoculated in the laser confocal culture dish, and when the degree of cell fusion reached 60%–70%, the cells were fixed, made transparent and blocked according to the instructions of the RNA-FISH detection kit (RiboBio, Guangzhou, China). Then, LncRNA Fish Probe Mix (RiboBio) was used for light-avoiding hybridization overnight at 37 °C; 18 S and U6 (RiboBio) were used as positive controls for the cytoplasm and nucleus, respectively. The nucleus was labeled with DAPI (RiboBio). PBS was used to wash the cells. Anti-fluorescence quenching agent (Solarbio) was then added, and the fluorescence was observed with a laser confocal microscope.

Fibroblasts were seeded on 20-mm microscopy culture dishes at approximately 1×10⁴ per well. Cells were fixed at room temperature for 20 minutes using 4% paraformaldehyde, then simultaneously blocked and permeabilized with a solution of 0.3% Triton X-100, 3% BSA, and 5% normal donkey serum (NDS) in phosphate-buffered saline (PBS) for 1 hour at room temperature. Cells were incubated with primary antibodies against IL-11 Rα (R&D Systems, Minneapolis, USA), α-SMA (Abcam, Waltham, USA), collagen type I α1 (COL1A1, Cell Signaling Technology, Beverly, USA) overnight at 4°C, and probed with the Alexa Fluor 488 or Alexa Fluor 546-labeled secondary antibody (both from Nanoprobes, New York, USA) in the dark at room temperature for 1 hour. Nuclei were visualized by staining with DAPI (Bioss, Beijing, China) for 10 min. Finally, an anti-fluorescence quenching agent (Solarbio, Beijing, China) was added, and samples were sealed and mounted. Samples were observed and imaged using a confocal microscope (Carl Zeiss 880, Oberkochen, Germany).

Immunofluorescence staining was performed as described in previous reports [32 (link), 33 (link)]. Briefly, the cells were grown on coverslips and treated for 3 days. After washing with PBS, the cells were fixed with 4% paraformaldehyde, permeabilized with PBS containing 0.1% (v/v) Triton X-100 (Solarbio), and incubated with 5% bovine serum albumin (Solarbio). After that, the cells were incubated with anti-TRPM7 (rabbit polyclonal antibody, 1:80, GeneTex, Irvine, CA) or anti-RUNX2 (rabbit monoclonal antibody, 1:500, Abcam) primary antibody at 4 °C overnight, followed by incubating with secondary antibody at room temperature for 1 h. The secondary antibody was DyLight 549 goat anti-rabbit IgG (1:200, Abbkine, Wuhan, China) or CoraLite488-conjugated goat anti-rabbit IgG (1:100, Proteintech, Rosemont, IL). If the cells needed microfilaments staining, they were incubated with Actin-Tracker Green (Beyotime) according to the manufacturer’s instructions. The cells were then counterstained with DAPI for 5 min and mounted with an anti-fluorescence quenching agent (Solarbio). The stained cells were observed under a laser scanning confocal microscope. The ImageJ 1.4.3.67, GraphPad Prism 7.0, and Origin 2019 (OriginLab Corporation, Northampton, MA) software packages were used for image processing.