SEM Sample Preparation: Hydrogel-Cell Complex Fixation and Imaging

Discard the spent media in the 48-well plate or remove the coverslip of the eCHIP. Carefully remove the hydrogel-cell complex or hydrogel using ophthalmic forceps, and cut it into cubes of approximately 1cm1cm1cm in size. Place the cubes in a microcentrifuge tube containing 1.5ml of 2.5% glutaraldehyde solution, and allow them to fix at room temperature for 1-2 hours before transferring them to 4°C overnight. Remove the fixative in a fume hood and rinse the samples three times with 0.1M PBS solution at pH 7.4. Aspirate the PBS solution and fix the samples with 1% osmic acid solution for 1.5 hours. Carefully remove the osmium solution in a fume hood and rinse the samples three times with 0.1M PBS solution at pH 7.4. Dehydrate the samples using a series of ethanol solutions with increasing concentrations (50%, 70%, 90%, 100%, 100%), then dry them in a critical point. Finally, sputter coat the ultrathin sections using a Q150T ES plus sputter coater (Quorum, England) and examine them using a Nova Nano SEM 450 at 80 kV (FEI, USA).