Ab209205

Five monoclonal antibodies anti-HLA-DQA1 antibody (1:10,000 dilution for WB, 1:200 dilution for IHC, ab128959), anti-GZMA antibody (1:1000 dilution, ab209205), anti-CD79A antibody (1:10,000 dilution, ab79414), anti-LTB antibody (1:1000 dilution, ab89568) and anti-HE5 antibody (1:1000 dilution, ab125071) were purchased from Abcam (Cambridge, UK). Monoclonal antibody anti-β-actin (1:20,000 dilution, 66009-1-Ig) was purchased from Proteintech (Wuhan, China).

The multispectral immunofluorescence (IF) staining method was performed as previously reported [38 (link)]. The samples were stained using the following antibodies: anti-CD8 (ZCIA055, zuochengbio), anti-CD57 (ZCIA273, zuochengbio), anti-GZMA (ab209205, Abcam), and anti-GSDMB (12885-1-AP, ThermoFisher). The samples were mounted using ProLong Diamond Antifade mounting medium containing DAPI (Invitrogen) and then sliced into 5 μm sections before being placed onto adhesive microscope slides. We performed deparaffinization, rehydration, and antigen retrieval to prepare the slides for multiplexed immunofluorescence staining. TG TSA Multiplex IHC Assay Kits (TissueGnostics Asia-Pacific Ltd.) were used along with a spectral library constructed from single-staining tissue images of each reagent to guide the staining process. The imaging procedure was performed utilizing the TissueFAXS (TissueGnostics) system, in conjunction with the Zeiss Axio Imager Z2 Microscope System, at a magnification of ×20. Quantitative analysis of the stained samples involved measuring cell density, expression levels, and area per cell using StrataQuest software (version 7.1.129, TissueGnostics GmbH, Vienna, Austria).