Histopathological Analysis of Gsdma3-Mutant Mice

Skin samples were harvested from the dorsal track of Gsdma3-mutant mice or C57BL/6 mice, and fixed in 4% paraformaldehyde in PBS for overnight. Sections were cut at 5 mm, dewaxed by xylene, and rehydrated by ethanol in gradient concentrations. For hematoxylin-eosin (H&E) staining, sections were stained with hematoxylin and eosin for 1 min, respectively, and then mounted with resinene. For immunostaining, sections were subjected to antigen retrieval with 0.1M citric acid buffer at ph6.0. Then the samples were incubated with primary antibodies against aSMA (1:100, Boisynthesis Biotechnology Co., Ltd. Beijing, China), Wnt5a (1:1000, Abcam, Cambridge, USA) and Gsdma3 (1:100, GL Biochem, Shanghai, China) [24 (link)] at 4°C for overnight. After wash, the samples were incubated with Alexa Fluor 594 (1:500, Beyotime, Jiang Su, China) conjugated secondary antibodies. Nucleus was labeled with 4’, 6’-diamidino-2-phenylindole (DAPI, 1: 1,000, Sigma-Aldrich, St. Louis, MO, USA).