Tissue specimens obtained from patients with CRS were immediately fixed in 4% paraformaldehyde and cut into coronal slices with a thickness of approximately 4 μm. The slices were stained with hematoxylin & eosin (HE) as previously described [23 (link)] and immunofluorescence (IF) was performed as previously described [14 (link)]. For IF, the slices were incubated with primary antibodies (Rabbit Anti-GSDMD-NT, AF4012, Affinity; Rabbit Anti-NLRP3, bs-10021R, Bioss; Rabbit Anti-cleaved Caspase-1, AF5418, Affinity; dilution: 1:200), then incubated with secondary antibodies (FITC labeled Goat Anti-rabbit IgG, ZF-0311, ZSGB-BIO; Alexa Fluor 594 labeled Goat Anti-rabbit IgG, ZF-0316, ZSGB-BIO; dilution: 1:50) at 24–26 ℃ in the dark for 60 min and finally stained with 4′,6-diamidino-2-phenylindole (DAPI, AR1176, Boster). The specimens were observed under a microscope (IX73, Olympus, Japan).
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