Gc305010

After deparaffinization and rehydration of paraffin-embedded tissue sections, they were blocked with 3% BSA blocking solution (cat. no. GC305010, Servicebio, China) at room temperature for 30 min. The sections were then incubated overnight at 4°C with primary antibodies: MAP4K4 (Rabbit, cat. no. 55247-1-AP, Proteintech, China), CXCL8 (Rabbit, cat. no. 17038-1-AP, Proteintech, China), TFDP2 (Rabbit, cat. no. 11500-1-AP, Proteintech, China), TNIK (Mouse, cat. no. 67948-1-Ig, Proteintech, China), CDKN2C (Rabbit, cat. no. AF0620, Affinity Biosciences, China). After washing the sections, they were incubated with secondary antibodies (cat. no. GB23303, Servicebio, China) for 50 min. Finally, the sections were rinsed with water, counterstained with hematoxylin, and mounted with a coverslip. The results were examined under an optical microscope (Nikon, Japan).

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.