Orius 1200a

C2C12 cells were cultured in 10-cm-diameter tissue culture plates and were allowed to grow until they reached 90% confluency. Then, the cells were transfected with PEI-cit/ASO/Ap nanocomplexes and synthetized by the CA technique following the steps aforementioned. After three hours, transfected cells were fixed for 45 min in 3% glutaraldehyde diluted in 0.12 M phosphate buffer (pH 7.4) containing 0.5% CaCl₂. Cells were scraped off culture tissue plates and centrifuged for 10 min at 7000 rpm and subsequently 10 min at 12,000 rpm. Afterwards, cellular pellets were rinsed in 0.12 M phosphate buffer, post-fixed in 2% osmium tetroxide for 3 h, dehydrated in acetone, and embedded in araldite (Durcupan, Fluka, Buchs, Switzerland). Ultrathin sections were examined with a JEOL EM-1011 (Tokyo, Japan) electron microscope. Micrographs were taken with a camera (Orius 1200A; Gatan, Pleasanton, CA, USA) using the DigitalMicrograph software package (Gatan, Pleasanton, CA, USA). Electron micrographs were processed using Adobe Photoshop CC 2021 (v. 22.3.0. Adobe Systems, San José, CA, USA).

Cells were fixed at 4 °C in a mixture of 2% PFA and 2.5% glutaraldehyde in 0.1 M phosphate buffer (PB), then scraped, pelleted, washed in PB and postfixed in 1% osmium tetroxide and 0.8% potassium ferrocyanide. Samples were dehydrated with acetone and embedded in Spurr resin. Ultrathin sections were obtained using an Ultracut E (Reichert), picked up on copper grids and observed with the JEM 1011 (JEOL) electron microscope, operating at 80 kV. Micrographs were taken with a camera (Orius 1200A; Gatan) using the DigitalMicrograph software package (Gatan). Electron micrographs were processed using Adobe Photoshop CS6 (v.13.0.1) (Adobe Systems).

To analyze the ultrastructure of the lysosomes and mitochondria of GCs, 3 animals of each karyotype were perfused with 3% glutaraldehyde in 0.1 M phosphate buffer pH 7.4 under deep anesthesia. Brains were removed and post-fixed overnight with the perfusion buffer. Then, 300 μm thick slices were obtained using a vibratome and small fragments of the GCL of the hippocampus were dissected. Tissue fragments were post-fixed with 2% osmium tetroxide, dehydrated in increasing concentrations of ethanol, and embedded in Araldite. Ultrathin sections mounted in copper grids were stained with lead citrate and uranyl acetate and examined using a JEOL 201 (Tokyo, Japan) electron microscope operated at 80 kV. Electron micrographs were recorded using a camera (Orius 1200A; Gatan, Pleasanton, CA, USA).