Bz x700 digital imaging microscope

In general, scleral cells were seeded and grown to 70% confluence in 4-well chamber slides (ThermoFisher) under 3 treatment groups and treated daily for 7 days: DEX (100 nM dexamethasone dissolved in DMSO [Millipore Sigma, Burlington, MA]), DMSO (0.1% [Millipore Sigma]), and MED (media). After 7 days, cells were washed with 1X phosphate buffered saline (PBS; Millipore Sigma), fixed with 4% paraformaldehyde for 25 minutes at room temperature (RT), washed 3 times with PBS, and blocked/permeablized with 0.25% Triton X-100 in 5% bovine serum albumin (BSA; Rockland, Limerick, PA) for 30 minutes at RT. Fixed cells were incubated in α-smooth muscle actin primary antibody (α-SMA; ab5694; Abcam, Cambridge, MA; 1:100) with 1% BSA overnight at 4°C. Cells were then washed twice with PBS, incubated in Texas Red-X secondary antibody (T-6391; ThermoFisher) with 0.2% BSA for 1 hour at RT, stained/mounted with DAPI (Vector Laboratories, Burlingame, CA), and imaged under a BZ-X700 digital imaging microscope (Keyence, Itasca, IL).

Scleral cells were grown to 70% confluence in 4-well chamber slides (ThermoFisher) under the same treatment groups (4 wells per group). After 7 days, cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature (RT) and blocked/permeablized with 0.25% Triton X-100 in 5% BSA for 30 minutes at RT. Fixed cells were incubated in phalloidin (Alexa Fluor 568 Phalloidin; ThermoFisher; 1:50) with 1% BSA overnight at 4°C, washed, stained/mounted with DAPI (Vector Laboratories), and viewed under a BZ-X700 digital imaging microscope (Keyence). Cells form two separate cell lines from two different donors were assessed.