Cy3 labeled goat anti rabbit igg antibody

A non-pathogenic strain of Candida bombicola (ATCC 22214 from Shanghai Bioresource Collection Center, China) was used to produce SL through a fermentation process, as described in a prior study^{25 (link)}. Histamine, loratadine, JNJ7777120 and capsaicin were obtained from Aladdin Biochemical Technology Co. Ltd. (Shanghai, China). HTMT and VUF8430 were purchased from Kuma Biochemical Technology Co. Ltd. (Shanghai, China). Fluo-4 AM, rabbit polyclonal TRPV1 antibody, Cy3-labeled goat anti-Rabbit IgG antibody and Hanks' Balanced Salt Solution (HBSS) were obtained from Beyotime Biotechnology Co. Ltd. (Shanghai, China).

Immunofluorescence staining was performed to detect the nuclear translocation of NF-κB p65 in KGN cells [38 (link)]. KGN cells grown on coverslips were fixed in 4% paraformaldehyde (Sinopharm Chemical Reagent, Beijing, China) for 15 min, incubated with 0.1% Triton X-100 (Beyotime Institute of Biotechnology) for 30 min and washed by phosphate buffer (PBS) for 3 times. Then, the cells were immersed in goat serum (Solarbio Science & Technology) at room temperature for 30 min and incubated with primary NF-κB p65 antibody (1:200, Proteintech Group) at 4°C overnight followed by secondary Cy3-labeled goat anti-rabbit IgG antibody (1:200, Beyotime Institute of Biotechnology) at room temperature for 1 h. After rinsing in PBS for 3 times, the cells were stained with 4ʹ,6-diamidino-2-phenylindole (DAPI, Beyotime Institute of Biotechnology), covered with mounting medium (Solarbio Science & Technology) and observed under a fluorescence microscope at a magnification of 400 × .