2.14 Tissue immunofluorescence staining
For tumor immunofluorescence staining, tissue sections were prepared
as described above. The sections were immersed in a Tris/EDTA buffer
(pH 8.0; Servicebio, Wuhan, China) and were heated for 15 min in a
microwave oven. After antigen retrieval, the sections were circled with
a PAP pen (Electron Microscopy Sciences, Fort Washington, PA, USA) to
reduce reagent waste. The sections were incubated with 3% BSA for 30
min at room temperature. Afterwards, the sections were incubated with
primary antibody at 4 °C overnight, washed three times with TBST, and
incubated with the corresponding FITC-conjugated secondary antibody
(Invitrogen Corporation, Carlsbad, CA, USA; RRID:AB_2535643; 1:500) for
50 min at room temperature in the dark. After washing with TBST for
three times, the sections were incubated with tissue autofluorescence
quencher for 5 min. Ultimately, the sections were stained with DAPI for
10 min. All stained slides were
mounted with Vectashield mounting medium (Vector
Laboratories, Burlingame, CA, USA). The fluorescence images were
captured by a fluorescence microscope (Leica, Wetzlar, Germany).