Blocking goat serum

The samples in OCT were cut into 30 μm sections on a cryostat microtome (MEV, SLEE Medical GMBH, Mainz, Germany) and then the sections were thaw-mounted on positively charged covered glass slides (18 × 18 mm, Citogtest scientific, Haimen, China). Immunofluorescence staining was performed according to the protocol. In brief, before staining, the sections were rinsed with phosphate-buffered saline (PBS) and processed with goat blocking serum (10%, 50 μL) (Solarbio life sciences, Beijing, China) for 1 h (room temperature). Then, the slides were incubated with TPRV4 primary antibody (1:500, 50 μL) (cat # ab94868, Abcam, Cambridge, UK) overnight (4 °C). After washing with PBS, the slides were incubated in the corresponding secondary antibodies (1:500, 50 μL) (cat # ab150081, Abcam, Cambridge, UK) for 1 h (room temperature). Subsequently, the slides were counter-stained with DAPI reagent (Thermo Fisher Scientific, Waltham, MA, USA) for 10 min (room temperature) and then washed again. Finally, the sections were sealed by anti-fluorescence quencher (Biosharp, Hefei, China) and examined under a fluorescence microscope (Axio Obesever Z1, Zeiss, Oberkochen, Germany).

After treatment, the cells were treated with 4% paraformaldehyde for 30 min, then blocked in 5% goat blocking serum (Solarbio, Beijing, China) for 30 min at room temperature. Primary antibodies were incubated at 4°C overnight. Full details on primary antibodies used are provided in Table 3. Then, the cells were incubated with goat anti-rabbit IgG (1 : 500; Multi Sciences, Hangzhou, China) secondary antibody for 2 h in the dark at 37°C. Finally, fluorescence images were captured with a fluorescence microscope (Olympus, Tokyo, Japan), and the analysis of the fluorescence images was performed by ImageJ.