IHC and IF were performed following standard procedures. Five-micron thick formalin-fixed paraffin embedded (FFPE) human tissue sections were used for the experiments. Stained slides were digitized using the Pannoramic DESK (3D HISTECH) with a 40× objective lens.
For IF, sections were stained with the Djc9 antibody (#NBP1–87903, Novusbio, 1:50) and CD20 antibody (#GB14030, Servicebio, 1:200) following the manufacturer’s instructions. Mean fluorescent intensity (MFI) was quantified using ImageJ (https://imagej.nih.gov/ij/ ). Colocalization analysis was performed using ImageJ plugin JACoP.
For IHC, sections were stained with the Djc9 antibody (#NBP1–87903, Novusbio, 1:500). Staining intensity score, nuclear staining intensity score, and staining percentage were quantified using ImageJ IHC Profiler plugin. Staining index was calculated as follows: staining intensity score (0–3) × staining percentage (0–100%). Staining intensity score was graded as follows: score 0: negative staining, score 1: weak staining, score 2: moderate staining, score 3: strong staining.
For IF, sections were stained with the Djc9 antibody (#NBP1–87903, Novusbio, 1:50) and CD20 antibody (#GB14030, Servicebio, 1:200) following the manufacturer’s instructions. Mean fluorescent intensity (MFI) was quantified using ImageJ (
For IHC, sections were stained with the Djc9 antibody (#NBP1–87903, Novusbio, 1:500). Staining intensity score, nuclear staining intensity score, and staining percentage were quantified using ImageJ IHC Profiler plugin. Staining index was calculated as follows: staining intensity score (0–3) × staining percentage (0–100%). Staining intensity score was graded as follows: score 0: negative staining, score 1: weak staining, score 2: moderate staining, score 3: strong staining.