Monochrome digital camera

Sterile scaffolds/ESF were placed in ultra-low attachment (non-tissue culture treated) (Thermo-Fisher scientific, Waltham, MA, USA) plates. Cells were added directly to the scaffolds/ESF at a seeding density of 1 × 10⁴ cells/scaffold and were allowed to attach for 2 h under 5% CO₂/air atmosphere at 37 °C. Afterwards, appropriate volumes of culture media were added to the wells and changed every other day for a total of 14 days. At the end of the incubation period, viable cells attached to the scaffolds were stained using the Calcein-AM from the LIVE/DEAD^® viability/cytotoxicity assay. Fluorescent images were taken using an inverted microscope with a monochrome digital camera using 5× magnification (Carl Zeiss Micro-Imaging, New York, NY, USA). Cell viability was assessed visually by estimating cell-covered areas compared to the control.

Immunofluorescence staining was performed on cultured cells. Cells were fixed in 4% PFA for 20 min and washed with PBS/0.01% Triton X-100 (PBST) and permeabilized with 0.2% Triton X-100 in PBS at room temperature for 5 min. Next, blocking was performed for 1 h using 5% normal goat serum in PBS. Following this step, the cells were stained for the HA-tag (1:1,000, Cell Signaling) diluted in PBST/1% normal goat serum at 4°C overnight. The next day, cells were washed with PBST, and incubated with goat anti-rabbit or goat anti-mouse 594 secondary antibodies (all at 1/500, Invitrogen) in PBST at room temperature for 2 h. Following this incubation, cells were washed with PBST three times for 10 min each and mounted in Vectashield mounting media with DAPI (Vector Labs, Burlingame, CA) and cover slipped for imaging. Fluorescence microscopy was performed using a Zeiss AxioImager M1 system equipped with epifluorescence filters, a Zeiss monochrome digital camera and AxioVision software. All images were processed in Adobe Photoshop for brightness/contrast, orientation and background correction to better illustrate staining patterns.