Ar0030

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).

The paraffin sections of cancerous and benign lesions were baked for 2 h at 60°C, deparaffinized with dimethylbenzene and hydrated in gradient ethanol. Tissue antigen retrieval was performed using citrate sodium buffer (pH 6.0) at 95°C for 15 min and cooled at room temperature for 30 min. Endogenous peroxidase was blocked with methanol containing 3% H₂O₂ for 10 min, then the slides were treated with 5% normal goat serum (SL2; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) for 30 min to limit non-specific binding. The anti-Axl rabbit polyclonal antibody (1:100; ab37861; Abcam, Cambridge, MA, USA) was overlaid on the sections and incubated overnight at 4°C. Following re-warming at room temperature for 1 h and three washes in PBS (AR0030; Wuhan Boster Biological Technology, Ltd., Wuhan, China), sections were incubated with horseradish peroxidase-labeled secondary antibody (1:500; SA00001-2; Wuhan Sanying Biotechnology, Wuhan, China) for 30 min at room temperature. Diaminobenzidine (DAB-0031; Fuzhou Maixin Biotech Co., Fuzhou, China) was used for antigen detection. Subsequently, the slides were counterstained with hematoxylin for 10 sec, dehydrated in gradient ethanol and mounted, and then viewed under an optical microscope. Negative controls used for comparison were incubated with phosphate buffer saline instead of the primary antibody.