2.7. Immunohistochemistry

After deparaffinization and rehydration, endogenous peroxidases were inhibited by incubation in 0.3% H₂O₂ in methanol for 20 min, followed by washing (5 min × 2) with distilled water. The samples were then spread onto slides, which were blocked with goat serum for 20 min at room temperature, followed by overnight incubation at 4 ℃ with an anti-GFAP antibody [1∶1000 in phosphate-buffered saline (PBS); ab7260, Abcam, Cambridge, UK]. The next day, the slides were rinsed with PBS (5 min × 3) and incubated with goat anti-rabbit IgG (H + L) secondary antibody (biotin conjugate; 1∶100 in PBS; BA1003, Boster, Wuhan, China) for 30 min at room temperature. Following this, the slides were rinsed with PBS (5 min × 3) and subsequently stained with diaminobenzidine (DAB) and hematoxylin. The slides were then examined and photographed using an Olympus BX53 microscope (Tokyo, Japan). The DAB staining intensity was analyzed using the Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, MD, USA) and expressed as integrated optical density values.