Tcs sp8 microscope system

In order to quantify changes in synaptic marker densities, we used the MAP protocol as previously described (Ku et al., 2016 (link)). In order to quantify changes in synaptic marker densities, we used the MAP protocol as previously harvested, embedded in hydrogel, and sliced into 100 μm thick (original thickness) transverse sections, which are then cleared, denatured, and immunostained. MAP-processed hippocampal slices were incubated with primary antibodies in PBS with 0.1% Triton X-100 (PBST) at 37 °C overnight, followed by washing at 37 °C for 2 hr in PBST three times. The tissue was then incubated with secondary antibodies in PBST at 37 °C overnight, followed by washing at 37 °C for 2 hr in PBST three times. Antibodies against pre- and post-synaptic markers (Bassoon and PSD-95) were used to label excitatory synapses. Finally, at least 1 hr before imaging, sections were linearly expanded 4 fold, preserving fine sub-cellular structures and protein localizations. With this expansion, high-resolution confocal z-stack imaging of synaptic marker localization was carried out using a Leica TCS SP8 microscope system with 63x, 1.30 NA glycerol-immersion objective (WD 0.3 mm).