Frozen sections of skin tissue up to 6 µm thick were prepared and fixed in pre-chilled acetone for 10 min, then washed three times with PBS for 10 min each and incubated for 30 min at room temperature with 0.5% Trition100 (50 µL of trition100 in 100 ml of PBS). Trition incubation was not required for immunofluorescence experiments with mTOR antibody (Abmart, T55306) in this study. PBS was washed three times for 10 min each and blocked with 1% BSA for 1.5 h. The antibodies (anti-AKT1 + AKT2 + AKT3:Abcam, ab179463, phospho-PI3-kinase p85-alpha/gamma:Abmart, T40116F, mTOR antibody:Abmart, T55306) were diluted in BSA and incubated overnight in a wet box at 4 °C. The antibodies were recovered and the sections were washed 3 times with PBS for 10 min, added with a fluorescent secondary antibody corresponding to the primary antibody, incubated for 1 h at room temperature in a wet box, washed 3 times with PBS, added with DAPI and photographed under a microscope (ECHO, RVL-100-G, USA).
Wang J., Hua G., Chen J., Cui K., Yang Z., Han D., Yang X., Dong X., Ma Y., Cai G., Zhang Y., Li J., Tai Y., Da L., Li X., Ma L., Ma Q., Li R., Liu J., Darwish H.Y., Wu K., Rong W., Liu W., Zhao Y, & Deng X. (2023). Epigenetic mechanism of Gtl2-miRNAs causes the primitive sheep characteristics found in purebred Merino sheep. Cell & Bioscience, 13, 190.
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