Immunofluorescence Staining Protocol

Hematoxylin–eosin (HE) and immunohistochemical staining were performed as previously described [19 (link)]. For immunofluorescence detection, the sections were routinely dewaxed and hydrated, and antigens were retrieved by microwave heating. Sections were treated with an autofluorescence quencher (Servicebio, Wuhan, China) and incubated with 5% (w/v) bovine serum albumin for 30 min at room temperature. Next, sections were incubated with primary antibody (1:100; Santa Cruz or 1:150, Proteintech) overnight at 4 °C. After being washed with PBS, the sections were incubated with FITC- or ALEX-conjugated goat anti-mouse IgG (1:200; Abcam, Waltham, MA, USA) at room temperature for 1–1.5 h in the dark. The nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). Finally, the antifluorescence quencher was used to seal the sections. Images were observed and obtained using a fluorescence microscope (LSM800, ZEISS, Oberkochen, Germany).