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).
Alexa fluor 594
Manufactured by Beyotime
Sourced in China
Alexa Fluor 594 is a fluorescent dye. It has an excitation maximum at 590 nm and an emission maximum at 617 nm, resulting in a bright red fluorescence. The dye can be used for various biological applications, such as labeling proteins, cells, or nucleic acids for fluorescence microscopy and flow cytometry.
Automatically generated - may contain errors
3 protocols using alexa fluor 594
1
Histopathological Analysis of Gsdma3-Mutant Mice
2
Cell Proliferation Assay with EdU Labeling
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U87-MG and U251 cells (5 × 104 cells/well) were seeded into 24-well culture plates. After incubation for 48 h, the cells were cultured with 50 µM EdU buffer for 2 h at 37 °C. The cells were fixed with 4% formaldehyde for 15 min and permeabilized with 0.3% Triton X-100 for 10 min. After washing with PBS two times, EdU solution with Alexa Fluor 594 (Beyotime, Shanghai, China) was added to each well. Nuclei were stained with DAPI Staining Solution (Beyotime, Shanghai, China). The results were visualized by a fluorescence microscope (IX71; Olympus Microscopes) at a magnification of × 200. The micrographs were analyzed by ImageJ software (version 1.8.0; National Institutes of Health).
3
Quantifying Cell Proliferation by EdU Labeling
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EdU labeled with Alexa Fluor 594 (Beyotime, China) was used to detect the DNA replication capacity of the transfected cells. The cells were cultured in a complete medium containing 10 μM EdU for 2 h in a cell incubator after washing with PBS 3 times. Then, the cells were fixed with 4% paraformaldehyde for 10 min and stained with DAPI for 5 min for nuclear staining. Finally, images of five random fields were captured at 100 × magnification under an Olympus fluorescence microscope (Tokyo, Japan). The intensity of the EdU staining was analyzed and calculated by ImageJ software and is presented as the fold change compared with the control.
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