Tissue Sampling and Preparation Protocol

After the completion of each experimental protocol, the brain, heart, lungs, kidneys, liver and pancreas were sampled and immersion-fixed in a solution containing 10% (w/v) neutral buffered formalin with gentle shaking. Sampling was performed by the same person at each experimental time point, randomly from the same region of each organ. After fixation, each tissue piece was processed and embedded into a paraffin block using the Excelsior tissue processor (Thermo Fisher Scientific). These paraffin blocks were trimmed on the Shandon Finesse 325 microtome (Thermo Fisher Scientific) to 5 μm sections. Sections were mounted on TruBond 380 adhesive slides and allowed to dry overnight at room temperature. All of the slides for the following tissue analysis were processed with deparaffinization and rehydration as previously described^{3 (link)}. In brief, the sections were deparaffinized in 2 changes of Histo-Clear II (64111–04, Electron Microscopy Sciences) for 10 min each. The slides were then transferred to 100% alcohol, for two changes, 10 min each, and then transferred once through 95%, 70% and 50% alcohol for 5 min each. The slides were then rinsed in water and washed in wash buffer (0.05% Tween-20 in 1× PBS) for 10 min.