Anti alpha smooth muscle actin α sma

Briefly, fibrin derived hydrogels with encapsulated hCSCs (12 µL) were prepared between cover slips to yield thin samples. The thin hydrogel samples were recovered at different time points and washed with PBS. After washing, the hydrogel samples were fixed with 3.7% formalin solution in PBS (Sigma-Aldrich, India) for 10 min at RT and washed with PBS again. Immunohistochemistry (fluorescence) was performed to visualize the expression of scarless-wound healing related markers. Primary anti-CD73, anti-CD90 and anti-alpha smooth muscle actin (α-SMA) antibody were procured from Abcam. Goat anti-mouse and anti-rat antibodies (conjugated with Alexa Fluor 594 and Alexa Fluor 488; Thermo Fisher Scientific) were used as secondary antibodies. Briefly, after washing, the hydrogel samples were blocked in BSA (5% in PBS, 1 h) and were incubated with primary antibodies at 4 °C overnight (1:100 dilution in 1% BSA). The samples were then washed (PBS, 3 times, 5 min each) and incubated with an appropriate secondary antibody (1:200 dilution in 1% BSA) at room temperature for 1 h. The sections were then washed (PBS, 3 times, 10 min each), semi dried, and mounted with Vectashield antifade mounting medium containing DAPI (#H1200, Vector Labs, Burlingame, CA, USA). The sections were imaged under a fluorescence imaging system (Evos FL Auto 2).

The cells prepared as described above were washed with PBS, fixed with 4% paraformaldehyde phosphate (PFA) for 10 min, and permeabilized with 0.2% Triton X-100 for 3 min. Subsequently, the sections were incubated for 24 h at 4°C with primary antibodies and incubated with fluorescein-conjugated secondary antibodies for 1 h at room temperature. The primary antibodies used were anti-collagen type I alpha 2 (COL1A2) (1:200, Abcam, Cambridge, UK), anti-fibronectin 1 (FN1) (1:200, Abcam), anti-alpha smooth muscle actin (αSMA) (1:200, Abcam), anti-p-Smad3 (1:200, Cell Signaling Technology, USA), anti-snail family transcriptional repressor 2 (SNAIL2) (1:200, Invitrogen, Carlsbad, CA, USA), and anti-zinc-finger-enhancer binding protein 1 (ZEB1) (1:200, Proteintech, Rosemont, IL, USA). Cover glasses were mounted using VECTASHIELD mounting medium with 4,6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA, USA).