Immunohistochemistry Staining Protocol

Tissues in the paraffin blocks were cut to a thickness of 4 μm and subjected to deparaffinization with xylene and dehydration with ethanol. For antigen retrieval, tissue sections were boiled in sodium citrate buffer (10 mM sodium citrate and 0.05% Tween 20, pH 6.0) for 10 min on a hot plate and then incubated at 25 °C for 20 min. After washing with PBS, the tissue sections were quenched in 3% H₂O₂ in PBS and incubated in blocking buffer (PBS, 1% BSA, and 0.5% Triton X-100) at 25 °C for 30 min. After washing with PBS, the tissue sections were incubated overnight with the primary antibody in a humid chamber at 4 °C. Tissue sections were washed with PBS and incubated with an HRP-conjugated secondary antibody (Invitrogen) in a humid chamber at 25 °C for 1 h. Tissue sections were washed with PBS, stained with 3,3-diaminobenzidine chromogen (Thermo) for 5 min at 25 °C, washed with distilled water, and counterstained with hematoxylin (DAKO, Carpinteria, CA, USA). Stained tissue sections were mounted using Permount (Thermo Fisher Scientific), and the stained images were examined by light microscopy. IHC staining was performed using the IHC profiler plugin of the ImageJ software [46 (link)].