Immunohistochemical (IHC) and immunofluorescence (IF) staining
For IHC staining, the deparaffinized sections were subjected to
heat-mediated antigen retrieval and blocked in
H2O2 for 30 min. Then, the sections were
blocked with 5% BSA at room temperature and stained overnight with the
following primary antibodies: anti-ACSL4 (1:500; Boster, Wuhan, China),
anti-GPX4 (1:500; Abmart, Shanghai, China), anti-xCT (1:500; Boster,
Wuhan, China), anti-IL-6 (1:500; Abclonal, A1570, Shanghai, China),
anti-IL-17 (1:200; Abclonal, A10587, Shanghai, China), anti-NLRP3
(1:300; Abclonal, A12694, Shanghai, China), anti-ASC (1:300; Abclonal,
A16672, Shanghai, China), anti-Caspase-1 (1:300; Abclonal, A0964,
Shanghai, China), anti-ASC (1:300; Abclonal, A16672, anti-IL-1β (1:300;
Proteintech, Wuhan, China), anti-F4/80 (1:300; Cell Signaling
Technology, Q61549, USA), anti-CD86 (1:300; Abclonal, A16805, Shanghai,
China), anti-iNOS (1:300; Abclonal, A3774, Shanghai, China), anti-CD206
(1:300; Cell Signaling Technology, Q61830, USA), anti-NF-κB (1:200;
Abclonal, A2547, Shanghai, China), anti-IKBα (1:200, Abclonal, A1187,
Shanghai, China), anti-p-IKBα (1:200; Abclonal, AP0707, Shanghai, China)
and anti-p-NF‑κB (1:200; Abclonal, AP0944, Shanghai, China). The
sections were incubated with a secondary antibody (1:5000; HRP, Donkey
Anti-Goat IgG, Proteintech) after being washed with PBS for 15 min at
room temperature. The colors were developed with a DAB kit (3,3′-
diaminobenzidine, Reith Biotechnology (Shanghai) Co., Ltd., China).
The cells were fixed with 4% paraformaldehyde (LAISI Biotechnology
(Shanghai) Co., Ltd., China) for 10 min, treated with 1% Triton-100 for
30 min, and washed twice with PBS. Then, the cells were blocked for 30
min with 5% BSA at room temperature. The cells were then incubated
overnight at 4 ℃ with the following primary antibodies: anti-ACSL4
(1:500; Abmart, Shanghai, China), anti-SLC7A11 (1:500; Abmart, Shanghai,
China), and anti-GPX4 (1:500; Abmart, Shanghai, China). The cells were
then incubated with secondary antibodies (1:500; HRP, Rabbit Anti-Goat
IgG, Proteintech, America) for 30 min and washed three times with PBS at
room temperature. Then, before coloration using a DAB kit
(3,3’-diaminobiphenyl, LAISI Biotechnology (Shanghai) Co., Ltd., China),
nuclei were stained with hematoxylin (LAISI Biotechnology (Shanghai)
Co., Ltd., China). Finally, a Carl Zeiss LSM710 microscope was used to
obtain images.
For IF staining, the sections were incubated with anti-NF‑κB (1:200;
Abclonal, A2547, Shanghai, China), anti-CD86 (1:300; Abclonal, A16805,
Shanghai, China), anti-iNOS (1:300, Abclonal, A3774, Shanghai, China),
anti-IL-17 (1:200; Abclonal, A10587, Shanghai, China) and anti-CD206
(1:300; Cell Signaling Technology, Q61830, USA) at 4 °C overnight after
being blocked (Reith Biotechnology (Shanghai) Co., Ltd., China). Then,
the sections were incubated with Alexa Fluor 594-labeled or Alexa Fluor
488-labeled secondary antibodies (1:100; Proteintech, Wuhan, China) for
2 h at room temperature after being washed with PBS three times. The
nuclei were stained with DAPI, and images were captured with a Carl
Zeiss LSM710 fluorescence microscope. The cells were fixed with 4%
paraformaldehyde (LAISI Biotechnology (Shanghai) Co., Ltd., China) for
10 min and treated with 1% Triton-100 for 30 min. After being blocked,
the cells were incubated with anti-NF‑κB (1:200; Abclonal, A2547,
Shanghai, China), anti-iNOS (1:300; Abclonal, A3774, Shanghai, China),
anti-CD86 (1:300; Abclonal, A16805, Shanghai, China), anti-ACSL4 (1:500;
Boster, Wuhan, China), anti-GPX4 (1:500; Abmart, Shanghai, China),
anti-xCT (1:500; Boster, Wuhan, China), and anti-IL-6 (1:500; Abclonal,
A1570, Shanghai, China) at 4 °C overnight. Then, the samples were
incubated with Alexa Fluor594-labeled (1:100; Proteintech, Wuhan, China)
and Alexa Fluor488-labeled secondary antibodies (1:100; Proteintech,
Wuhan, China) at room temperature for 2 hours. The nuclei were
visualized with DAPI, and images were captured using a Carl Zeiss LSM710
fluorescence microscope.