Anti α sma antibody

Mice were euthanized and corneas excised. Whole-mount corneas were fixed in acetone and incubated in blocking solution containing 5% BSA and 0.5% Triton X-100 in PBS for 1 hour at room temperature. Corneas were washed in PBS and were incubated in blocking solution containing 10 µg/mL anti-α-SMA antibody (53-6496-82; Thermo Fisher Scientific) or control (53-4714-80; Thermo Fisher Scientific) overnight at 4°C. After they were washed in PBS, the corneas were mounted on glass slides using a 4′,6-diamidino-2-phenylindole (DAPI)–containing mounting medium. Slides were then visualized with a Leica SP8 confocal microscope (Leica, Wetzlar, Germany).

Formalin-fixed liver tissues were embedded in paraffin wax, sectioned transversely (5 μm), and stained with Hematoxylin & Eosin (H&E) and Masson's trichrome (MT) stains. The analyses were performed microscopically (Leica Imaging Systems, Cambridge, UK). At least 5 different fields of view were examined per each liver specimen. The degree of hepatic fibrosis was evaluated on a 0–6 scale, as previously described [53 (link)]. Moreover, the degree of portal inflammation (0–4), focal necrosis and inflammation (0–4), confluent necrosis (0–6), and periportal/periseptal interface hepatitis (0–4) were semiquantitatively assessed to provide a necroinflammatory activity score for each liver specimen (0–18) [53 (link)]. Moreover, the hepatic expression of α-smooth muscle actin (α-SMA) protein was detected immunohistochemically using a mouse monoclonal anti-α-SMA antibody (catalog number MS-113-R7, Thermo Fisher Scientific, UK). Three to five non-overlapping fields were analyzed using ImageJ software (version 1.53t, National Institutes of Health) to quantify the average percentage area of positive immunostaining for α-SMA in each liver specimen, as reported previously [54 (link)]. The pathologist performing histopathological assessments was unaware of the group assignment.