Cy3 tyramide

For assessment of CD8 T‐cell activity, immunofluorescence was performed on paired pre‐ and post‐treatment tumour samples from 12 patients (six responders and six non‐responders). Briefly, slides were deparaffinized and rehydrated before antigen retrieval. Endogenous peroxidase activity was blocked with 3% H₂O₂ at room temperature for 15 min. The slides were then incubated with a rabbit recombinant anti‐CD8 alpha antibody [CAL66] (ab237709, 1:1000; Abcam) overnight at 4°C, followed by horseradish peroxidase‐conjugated goat anti‐rabbit IgG at room temperature for 50 min in dark conditions. The slides were subsequently stained with CY3‐Tyramide (red, G1223, Servicebio). The nucleus was counterstained with DAPI (blue, G1012, Servicebio).

Paraffin-embedded DLBCL sections confirmed by pathologists were collected. Paraffin-embedded DLBCL sections were deparaffinized with the dewaxing agent and absolute ethanol. After that, citric acid solution (Servicebio, G1202) was used for antigen retrieval and 3% H2O2 was used to inactivate endogenous peroxidase. Then 3% bovine serum albumin (Servicebio, GC305010) was added to block sections for 30 minutes. Add the primary antibody, anti-CD79A (ZEN BIO, R23860), and incubate overnight at 4 °C. After washing sections three times using PBS, add goat anti-rabbit IgG H&L (HRP) (Servicebio, GB21303) and incubate for 50 minutes. After washing three times using PBS, add CY3-tyramide (Servicebio, G1223) for 10 minutes. Wash with a citric acid solution to remove the first type of primary antibody. Then add the second primary antibody, anti-HMGB3 (Affinity Biosciences, AB_2841269), and incubate overnight at 4 °C. After washing sections three times using PBS, add anti-rabbit IgG (Alexa Fluor 488 conjugate) (Servicebio, GB25303) and incubate for 50 minutes. Finally, sections were stained with DAPI (Servicebio, G1012) and sealed with an anti-fluorescent quenching agent (Servicebio, G1401). Finally, fluorescence image capture was performed using laser scanning confocal microscopy and processed using ImageJ software.