Immunohistochemical analysis of GSDMD and p-p65 in brain sections

Brain paraffin sections (4 μm) were hydrated and Tris/EDTA buffer performed heat-mediated antigen retrieval for 20 min. The sections were then processed with 3% H₂O₂ for 10 min. The sections blocked with 5% BSA for 1 h were incubated with primary antibodies GSDMD (Abclonal), phosphorylated p65 (Abclonal) overnight at 4°C, and then incubated in HRP-labeled secondary antibodies (Proteintech). DAB (Servicebio) was utilized for dyeing while hematoxylin was used for nuclei staining. Images were acquired using the Olympus BX53 microscope (Olympus). The distribution and intensity of GSDMD and p-p65 staining was described by a semiquantitative score in a blinded fashion (0-negative, 1-weak, 2-moderate, 3-strong, and 4-strong and widely distributed) (n = 6/group) (Xu et al., 2020 (link)).