Dab peroxidase substrate kit

Liver tissue embedded in paraffin was processed for deparaffinization and rehydration. The tissue sections were then treated with 10 mM citrate buffer (pH 6.0) and subjected to antigen retrieval using microwave heating for 3 min. Subsequently, the sections were incubated with 3% hydrogen peroxide solution at room temperature for 20 min to block endogenous enzymes. Blocking was carried out using serum from the same source as the secondary antibody, incubating at 37°C for 30 min. The sections were then incubated overnight at 4°C with an anti-Tnfα antibody (Bioss, bs-10802R) diluted to 1:500 in the serum corresponding to the blocking solution. On the following day, the sections were incubated with an HRP-conjugated goat anti-rabbit IgG secondary antibody, diluted to 1:2000, at room temperature for 1 h. Subsequently, the sections were processed using the DAB Peroxidase Substrate Kit (MXB Biotechnologies, DAB-4033) and stained with hematoxylin. The stained sections were imaged and scanned using a 3D digital slide scanner (3D HISTECH) and analyzed using ImageJ software.