2.11 | Immunohistochemical and Immunofluorescence Staining
Immunohistochemical staining was performed for cleaved-caspase-3, pMLKL, and GSDMD-N. Briefly, paraffin sections (4 mm) were fixed in 3% formaldehyde and stained with cleaved-caspase-3 (BF0711, 1:50; Affinity), pMLKL (AF7420, 1:50; Affinity), and GSDMD-N (DF13758, 1:50; Affinity) primary antibodies. Goat anti-rabbit IgG and goat anti-mouse IgG were used as secondary antibodies. All sections were examined under a light microscope (Pannoramic Scan, 3DHISTECH) and digitized using a high-resolution camera.
The sections were blocked with a 10% bovine serum albumin (BSA) solution to avoid non-specific staining. The sections were incubated with cleaved-caspase-3 (BF0711, 1:200; Affinity), pMLKL (AF7420, 1:200; Affinity), and GSDMD-N (DF13758, 1:200; Affinity). Sections were incubated overnight at 4 °C, followed by incubation with goat anti-rabbit IgG (M21014; Abcam) and goat anti-mouse IgG (M21013; Abcam) antibodies and nuclear staining with 4′,6-diamidino-2-phenylindole (DAPI; Beyotime Biotechnology, Shanghai, China). The stained sections were examined using an inverted fluorescence microscope (CYTATION 5, BioTek, USA).