Ultrastructural Analysis of Mouse Hippocampal Mitochondria

For TEM, mouse brains were perfusion-fixed in 4% PFA and 50-μm sections were obtained by vibratome sectioning. The slices to be analysed were stored in 0.1 M PB until further processing. The slices were then transferred to polypropylene 24-well plates (Caplugs Evergreen) and processed using a PELCO BioWave Pro+ microwave (Ted Pella Inc.) and following a protocol adapted from the National Centre for Microscopy and Imaging Research protocol (Deerinck et al, 2010 (link)), which was detailed in Eder et al (2020 (link)). After processing and embedding of slices into Durcupan ACM resin (Sigma-Aldrich), the blocks were trimmed to a trapezoid around the hippocampus CA1 area. The samples were then sectioned using a UC7 ultramicrotome (Leica Microsystems), and 70-nm sections were picked up on Formvar-coated G50HEX copper grids (Gilder Grids Ltd.). The sections were viewed using a 120-kV Tecnai G2 Spirit TEM (FEI Company), and images were captured using an Orius CCD camera (Gatan Inc.). 22 control cells and 16 NDR1/2 knockout cells from respectively six and three different areas of the CA1 region were imaged at various magnifications. 16,500× images were randomised and anonymised using Advanced Renamer, for unbiased analysis of the mitochondrial morphology.