Immunohistochemistry and multiplex immunohistochemical staining
tumor
TUMOR, AMPLIFICATION, SECONDARY
A retrospective study was performed on formalin-fixed, paraffin-embedded (FFPE) tumor tissues of NPC and metastatic liver, lung and lymph gland. Multiplex immunohistochemical staining was performed as follow steps, 4 μm sections from full FFPE blocks of tumor tissues were sectioned, dewaxed, and fixed with 10% neutralized formaldehyde. Then, antigen was retrieved using heated Tris- EDTA buffer (pH 8.0 or pH 9.0) for 2.5 min. Each section was subjected to four successive rounds of antibody staining after the initial establishment of staining conditions for each individual primary antibody and successive optimization. Each staining step consisted of blocking with 20% normal goat serum/fetal bovine serum in PBS and incubation with primary antibodies, followed by biotinylated anti-mouse/anti-rabbit secondary antibodies and streptavidin–horseradish peroxidase (HRP) substrate. Then, the immunoreactive stains were visualized using tyramide signal amplification (TSA) with fluorophores Opal 480, 520, 570, and 690 diluted in 1×Plus Amplification Diluent. Finally, the Ab–TSA complexes were stripped in heated Tris-EDTA buffer (pH 8.0 or pH 9.0) for 2.5 min. Nuclei were counterstained with 4′, 6- diamidino-2- phenylindole, dihydrochloride (DAPI) and sections were mounted using Perma Fluorfluorescence mounting medium (PANOVUE). The same procedure without primary antibodies was used as a negative control. The multiplex immunohistochemical staining results were scored based on the percentage of the number of cell subsets. Computer recognition software used indicated molecules to identify subsets of cells automatically and count them. Cell quantification was performed across whole tumor sections using Halo analysis software (PANOVU). The antibodies used for IHC staining are CD4 (ZM-0418, ZSbio, Clone: EP204); CD19 (ZM-0038, ZSbio, Clone: UMAB103); CD8 (ZA-0508, ZSbio, Clone: SP16); CD3 (ZM-0417 ZSbio Clone: LN10); Eomes (ab183991, Abcam, 1:200); α-SMA (ab7817, Abcam, 1μg/mL); KDR (ab2349, Abcam, 1:100); PD-L1 (ab205921, Abcam, 2μg/mL); CD31(ab28364, Abcam, 1:50); IL8 (94407 T, Cell Signaling Technology, 1:100); VEGFA (ab52917, Abacm, 1:100); c-KIT (ab32363, Abcam, 1:400); SRC (ab109381,Abacam, 1:400).