Immunofluorescence staining was performed on both lung tissue and cellular slices through routine operation. For tissue staining, after dewaxing, rehydrating, antigen repairing, and blocking, paraffin-embedded tissue slices were incubated with diluted primary antibodies of anti-F4/80 (#GB11027, Servicebio), anti-Cytokeratin 7 (CK7, #GB11225, Servicebio), and/or anti-ADAR1 (#SC-73408, Santa Cruz), anti-cleaved-GSDMD (#AF4013, Affinity, Jiangsu, China), TUNEL (#GDP1043, Servicebio), anti-iNOS (#GB11119, Servicebio), or anti-CD163 (#GB11340-1, Servicebio) at 4 °C overnight, followed by incubation with their corresponding secondary antibodies at room temperature for 1 h in the dark. Between double staining procedures, 488-TSA (Servicebio) incubation and reantigen repair were conducted. DAPI (#G1012, Servicebio) was used for nuclear counterstaining. The slices were mounted with antifluorescence quenching sealing reagents (Boster, Wuhan, China) and captured under a fluorescence microscope (Life Technologies, Carlsbad, USA). For cellular staining, the primary antibodies used were anti-cleaved GSDMD (#AF4013, Affinity) and anti-caspase-1 (#GB11383, Servicebio). Mean fluorescence intensity (MFI) in a random field of view was measured using ImageJ software.
Zhao X., Xie J., Duan C., Wang L., Si Y., Liu S., Wang Q., Wu D., Wang Y., Yin W., Zhuang R, & Li J. (2024). ADAR1 protects pulmonary macrophages from sepsis-induced pyroptosis and lung injury through miR-21/A20 signaling. International Journal of Biological Sciences, 20(2), 464-485.
+ Open protocol