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).