Morgagni 268

Monolayers of human hiPSC-CMs were fixed in 2% (v/v) glutaraldehyde in 0.1 M HEPES buffer (pH 7.4) for 20 min, washed in DPBS and stored in 1% (v/v) glutaraldehyde in DPBS until analyses. Samples were washed, contrasted, dehydrated, infiltrated, embedded, cured, dissected into small blocks and mounted on epon dummy blocks, as previously described^{34 (link)}. Ultrathin sections of cell monolayers were cut on a Leica UC6 ultramicrotome using a diamond knife. Sections were collected on formvar-coated slot grids, stained with lead citrate and uranyl acetate as described in the literature^{35 (link)}, and analyzed on a FEI Morgagni 268 at 80 kV. Images were taken with an Olympus MegaView III using the iTEM software.

The surface morphology of the freeze-dried optimized rivastigmine nanoparticles were studied for their shape, surface microstructure (porous/hollow/aggregated mass and individual particle sizes were determined using SEM (Leo Corporation, Zeiss leica, UK), and TEM (Morgagni 268, Olympus).

Fifty microliter of nanoparticles dropped in the parafilm and copper grid were kept above the samples and waited for 1 min. Then, the copper grid was taken out and dropped in the 2% phosphotungstic acid, and waited for 30 s. Afterwards, copper grid was taken out and dried with help of tissue paper, and then placed under the TEM microscope (Morgagni 268, Olympus). Pictures were taken at various magnifications (× 13,000), and accelerating voltage 70 kV and data were analyzed using Olympus soft imaging viewer.