Ab9475

Four-micrometre sections of FFPE tissue from patients with OSCC were subjected to immunohistochemical staining. Before staining, all specimens were incubated at 60 °C for 2 h. Then, the samples were deparaffinized in xylene, rehydrated in graded alcohol baths, and incubated in 3% H₂O₂ for 20 min. Heat-induced antigen retrieval was performed in 0.01 M sodium citrate buffer at pH 6.0. Prior to antibody incubation, the slides were placed in a microwave and heated for 10 min at low temperature for antigen retrieval. Specimens were then incubated in 3% BSA for 30 min with the following primary antibodies: rabbit anti-CD20 (1:50, ab9475, Abcam, Cambridge, UK), rabbit anti-CD3 (MAB-0740, MXB Biotec, Fuzhou, China) and rat anti-PNAd (1:100, 120802, BioLegend, San Diego, CA, USA) overnight at 4 °C. Then, the cells were incubated with the secondary antibody at 37 °C for 30 min, stained with DAB for 3–8 min according to the primary antibody, and counterstained with haematoxylin.

Paraffin sections from the 200 cases of GC patients were dewaxed in xylene and ethanol. After cleaned in distilled water, the paraffin sections were pretreated with citrate buffer, pH 6.0 (CD177) and EDTA Antigen Retrieval Solution, pH 8.0 (CD4, CD8, CD20, CD56, CD68, and CD117) for 3 min at 120°C in a pressure cooker, and endogenous peroxidase was inhibited with 3% H₂O₂ in PBS for 10 min. Nonspecific actions in the sections were also blocked with goat serum (BOSTER, USA) for 1 h at room temperature. The sections were then incubated with the primary antibody overnight at 4°C, followed by incubation with the secondary antibody for 30 min at 37°C. Primary antibodies used were CD4 (ab183685, 1 : 1000, Abcam, Cambridge, MA, USA), CD8 (ab4055, 1 : 100, Abcam, Cambridge, MA, USA), CD20 (ab9475, 1 : 50, Abcam, Cambridge, MA, USA), CD56 (ab75813, 1 : 100, Abcam, Cambridge, MA, USA), CD68 (ab213363, 1 : 4000, Abcam, Cambridge, MA, USA), CD117 (ab32363, 1 : 400, Abcam, Cambridge, MA, USA), and CD177 (ab220279, 1 : 200, Abcam, Cambridge, MA, USA). Second antibodies used were goat anti-rabbit IgG (CD4, CD8, CD56, CD68, CD117, and CD177) and goat anti-mouse IgG (CD20). The chromogenic reaction was performed via diaminobenzidine (DAB) staining, and the staining intensity was measured using Image-Pro Plus version 6.2 software (Media Cybernetics, Rockville, Maryland, USA).