α smooth muscle actin

A human mesangial cell line (HMC cell line, FH0241) was obtained from FuHeng Biology Co. (Shanghai, China). DHT (116064-77-8, purity ≥ 98%) was acquired from Krre Technology Co. (Beijing, China). Mannitol (69-65-8) was obtained from Macklin Biochemical Co. (Shanghai, China). The following antibodies: GAPDH (1:1000, ab8245), α-smooth muscle actin (1:1000, ab7817), Collagen I (1:1000, ab260043), Fibronectin (1:1000, ab2413), PI3KCA (1:1000, ab40776), PI3K (1:1000, ab191606), p-PI3K (1:1000, ab182651), AKT (1:1000, ab179463), and p-AKT (1:1000, ab192623) were bought from Abcam Co. (Cambridge, United Kingdom). Goat Anti-Rabbit IgG H&L (HRP, A0208), Rabbit Anti-Mouse IgG H&L (HRP, A0216), and Rabbit Anti-Mouse IgG H&L (Alexa Fluor 488, A0428) were purchased from Beyotime (Shanghai, China). DAPI Staining Solution (C1006) and Cell Counting Kit-8 (C0038) were also obtained from Beyotime (Shanghai, China). Glucose, Trypsin, Dulbecco’s Modified Eagle’s Medium (DMEM), and fetal bovine serum (FBS) were purchased from GIBCO. The Mouse Albumin ELISA Kit (ab207620) was purchased from Abcam.

Mouse eyeballs were enucleated and immediately fixed in 4% paraformaldehyde at 4°C overnight as previously described (Fu et al., 2018b (link)). After rinsed three times with PBS, the eyes were dehydrated and embedded in paraffin (Paraplast, Sigma-Aldrich, USA). Tissue slices (5 μm) were obtained using a rotation microtome (Thermo Fisher, USA), deparaffinized, and then rehydrated with graded ethanol for 5 min twice each. Antigen retrieval was conducted in citrate buffer. Once cooled, tissue sections were blocked with 10% goat serum and 0.2% Triton-X 100 in a dark and humid chamber for 2 h. After rinse briefly with PBS, the sections were immunolabeled with rabbit polyclonal antibody (α-smooth muscle actin, 1:100, Abcam) and incubated at 4°C overnight. After flushing, the samples were incubated with corresponding secondary antibodies (Alexa goat anti-rabbit 568, 1:500, Thermo Fisher) for 2 h. DAPI (Vector, CA) was used to visualize cellular nuclear. The slices were examined by the Keyence all-in-one fluorescence microscope (Itasca, USA) (Kasetti et al., 2016 (link)). For H&E staining, the paraffin section of mice TM tissues were sequentially deparaffinized, rehydrated, stained with hematoxylin and eosin (Sigma-Aldrich), dehydrated and sealed. The slices were visualized and photographed with phase contrast microscope (DMI 1, Leica).