Phosphate buffered saline (pbs)

Confocal microscopy was performed to visualise the morphology of the cells attached onto the scaffolds cultured in basal medium after 7 and 14 days. Each scaffold was rinsed twice in PBS (Polysciences, Warrington, PA, USA) and fixed in 4% (w/v) paraformaldehyde in PBS with 1% (w/v) sucrose at 37 °C for 4 h. Thereafter, the scaffolds were rinsed twice with PBS and then the samples were incubated 5 min with Triton 0.1% (w/v) at room temperature to permeabilise the cells. Then, the scaffolds were rinsed with PBS and incubated with 400 μL, 6-diamidino-2-phenyl-indole (DAPI) (DAPI, Vector Laboratories, Burlingame, CA, USA) (1:1000) and Atto 565-conjugated phalloidin at a concentration of 0.165 μM (Molecular Probes) for 30 min. DAPI (cell nuclei) and phalloidin (cytoskeleton) were visualised in blue and red, respectively.

Cover slips were chemically activated to allow stable, covalent formation of polyacrylamide sheets according to the protocol of Pelham and Wang (Pelham and Wang, 1998 (link)), with modifications (Davis et al, 2012 (link)). Briefly, a glass cover slip (No. 1, 15 mm diameter; Fisher Scientific, Pittsburgh, PA) was coated with a small drop of 3-aminopropyltrimethoxysilane (TESPA, Sigma, St. Louis, MO), which was spread evenly on the surface. After 5 minutes, the coverslips were washed extensively with distilled water and then were autoclaved (121°C at 1.5 atm) for 1 hour. The coverslips were transferred, treated side up, into plastic petri dishes and covered with 0.5% glutaraldehyde in phosphate buffered saline (PBS) (prepared by diluting 1 part of 70% stock solution, Polysciences, Inc., Warrington, PA, with 140 parts of PBS). After incubation at room temperature for 30 minutes, the coverslips were washed 5 times in distilled water on a shaker, for 10 minutes per wash, and allowed to dry in air. The treated coverslips were stored in a petri dish for up to 48 hours after preparation.