2.3. HMGB1 Immunohistochemistry

For HMGB1 detection, the samples were dewaxed in xylene and dehydrated in a graded ethanol series. Endogenous peroxidase activity was inhibited by incubating the slides with 0.3% H₂O₂ for 5 min, followed by washing thrice with PBS; the sections were incubated with the primary antibodies to HMGB1 (1 : 1000 dilution, ab18256; Abcam, Cambridge, UK) and incubated at 4°C overnight, washed in PBS, and incubated at 37°C for 1 h with biotinylated anti-rabbit/rat IgG (1 : 200; Maixin-Bio, Shanghai, China) according to manufacturer's instructions. The tissue was incubated with Streptavidin Peroxidase (Maixin-Bio) reagents at 37°C for 30 min, stained with freshly prepared DAB (Maixin-Bio). Morphometric quantification of the stained sections was performed with a customized digital image analysis system (IMAGE-Pro plus 4.5). Analysis of the kidney and capturing images was performed.