Dab chromogen

For HE assay, the slices of tumor tissues were stained with hematoxylin for 5 min, then washed for 1 min, and restored to blue by 1% ammonia (30 s). Then, slices were swashed with running water (1 min). After, slices were stained by 0.5% HE or 1 min, flushed for 30 s then transparentized and finally mounted with neutral gum. For IHC assay, the appropriate antibody diluent and antigen repair treatment methods were selected following the manufacturer’s instructions. The paraffin-embedded tissue was dewaxed in xylene, dehydrated in ethanol, and 3% H₂O₂ blocked for 10 min. The antigen was extracted in 95°C EDTA buffer for 15–20 min. Sections were incubated with Ki67 antibody (Santa Cruz, USA) overnight at 4°C. After washing 3 times with PBS, sections were incubated with the secondary antibody coupled to HRP (Santa Cruz Inc., USA) at 37°C for 50 min. Finally, DAB chromogen (Beyotime Inc., China) was added, and then slices were washed with water and stained with hematoxylin.

The expression of A20 and RIPK3 in paraffin-embedded tissue sections was analyzed by immunohistochemistry (IHC). Following deparaffinization and antigen retrieval in 10 mM sodium citrate buffer (pH 6.0) using the pressure cooker method at full power for 4 min, the tissue sections were exposed to 3% H₂O₂ for 10 min. The tissue sections were then blocked for 30 min at room temperature with 5% bovine serum albumin (BSA). The sections were incubated with anti-A20 (1:40) and anti-RIPK3 (1:200) antibodies overnight at 4°C in a humid chamber and then incubated with the horseradish peroxidase (HRP)-conjugated secondary detection antibody for 30 min at 37°C. The sections were then incubated with DAB chromogen, counterstained with hematoxylin (Beyotime Institute of Biotechnology) and finally dehydrated, cleared and mounted with neutral gum. The sections were washed with several changes of Tris-buffered saline (TBS)-0.3% Tween buffer between each step.