Abs117811

Protein samples from AD and normal tissues were added to sodium dodecyl sulfate-polyacrylamide gel (15 μg sample per gel lane) to separate, and then, the protein bands were transferred to polyvinylidene fluoride membranes. The membranes were blocked, followed by incubation with rabbit anti-YTHDC1 (abs117811, Absin, Shanghai, China) and rabbit anti-GAPDH (D110016, Sangon Biotech, Shanghai, China). The blots were then incubated with goat anti-rabbit IgG which was labeled by horseradish peroxidase. Finally, the density of immunoreactive bands normalized to the signal intensity of GAPDH was determined using the Electro Chemical Luminescence Kit and ImageJ software 1.53.

IF staining analysis of YTHDC1 and endothelial cell marker CD31 was carried out for cellular localization in AD tissue. AD tissue sections were passed through xylene, isopropanol, and ethanol for dewaxing and rehydrating. Dehydrated sections were placed in citrate buffer under high temperature and pressure for antigen retrieval. Then, add hydrogen peroxide to block endogenous peroxidase. After incubation, AD sections were blocked by Immunol Staining Blocking Buffer (P0102, Beyotime, Shanghai, China). Incubate tissue with rabbit anti-YTHDC1 antibody (abs117811, Absin, Shanghai, China) and the Cy3 (red)-conjugated goat anti-rabbit IgG (GB21303, Servicebio, Wuhan, China). Then, rabbit anti-CD31 antibody (abs120102, Absin, Shanghai, China) and the 488 (green)-conjugated goat anti-rabbit IgG (abs20025, Absin, Shanghai, China) were used to incubate the tissue sequentially. Finally, the sections were stained by Antifade Mounting Medium with DAPI (P0131, Beyotime, Shanghai, China). The prepared sections can be viewed under a fluorescence microscope.