Sc 66941

After 14 days of culture, samples were fixed, permeabilized and blocked using the same method used to prepared cells for F-actin staining. For immunocytochemical staining, samples were then incubated for 18 h at 4°C in a range of antibodies, anti-ALDH3A1 1:50 (ab76976; Abcam, Cambridge, United Kingdom), anti-keratocan 1:50 (sc-66941; Santa Cruz, Heidelberg, Germany), or anti-α-smooth muscle actin (αSMA) 1:50 (ab7817; Abcam). They were then washed thrice with PBS, 20 min for every wash and then incubated with fluorescently labelled antibodies for 1h at ambient temperature, in the dark. To highlight ALDH3A1 and Keratocan, Donkey anti-rabbit Alexa Fluor 488 (ab150073; Abcam) was used. For αSMA, goat anti-mouse biotin followed by ExtrAvidin-FITC (B7151 and E2761; Sigma). Constructs were washed with PBS and counterstained with DAPI (1 mg/mL,1:500 dilution) to visualize nuclei. Confocal microscopy was performed on the Leica SP8 with LAS X Advanced software 2.13 post-processing.

Cells were cultured on μ-slides (Ibidi GmbH, Martinsried, Germany). Wells were washed briefly in phosphate buffered saline (PBS) and fixed in 4% (wt/vol) paraformaldehyde (PFA) for 15 minutes at room temperature (RT). Cells were incubated with blocking buffer consisting of 2% (wt/vol) bovine serum albumin in PBS for 30 minutes at RT, and incubated with anti-keratocan 1:50 (sc-66941; Santa Cruz, Heidelberg, Germany), anti-ALDH3A1 1:50 (ab76976; Abcam, Cambridge, United Kingdom), and anti-α-smooth muscle actin (αSMA) 1:50 (ab7817; Abcam) at 4°C for 18 hours. Cells were rinsed in PBS and incubated with fluorescently labelled antibodies for 1 hour at RT in the dark. Keratocan and ALDH3A1 were detected with donkey anti-rabbit AlexaFluor 488 (ab150073; Abcam) and αSMA was detected with goat anti-mouse biotin followed by ExtrAvidin-FITC (B7151 and E2761). Cells were rinsed in PBS and incubated with 1 mg/mL 4′, 6′-diamidino-2-phenylindole 1:500 (DAPI) for nuclear staining. Filamentous-actin (f-actin) staining was performed using Phalloidin-TRITC 1:50. Samples were photographed using an inverted epifluorescent microscope coupled to a digital camera (Olympus IX71; Olympus).