Figure 4.
GA improves ultrastructure in both mouse and marmoset brain slices cleared with ScaleSF
(A) TEM images of mouse cerebral cortex cleared with ScaleSF (A1–A5) or stored in PBS(−) (A6, A7). Mouse brains were fixed with 4% PFA (A1, A6) or 4% PFA containing GA (0.02%, A2; 0.2%, A3; 1%, A4, A7; 2%, A5). Arrowheads indicate postsynaptic membranes. Brain slices were cleared with ScaleSF and embedded in ScaleS4 gel for 24 hr, or stored in PBS(−) at 4°C in the analysis. Scale bar, 500 nm.
(B) Scoring of membrane continuity of presynaptic terminals for each condition in (A). Over 90%, 50%–90%, 10%–50%, and less than 10% membrane continuity of presynaptic terminals are scored as 4, 3, 2, and 1, respectively (n = 31 synapses, GA 0%, ScaleSF; n = 52 synapses, GA 0.02%, ScaleSF; n = 33 synapses, GA 0.2%, ScaleSF; n = 34 synapses, GA 1%, ScaleSF; n = 31 synapses, GA 2%, ScaleSF; n = 32 synapses, GA 0%, Control [PBS-stored slices]; n = 31 synapses, GA 1%, Control [PBS-stored slices]; n = 3 mice for each condition; H = 52.44, df = 6, P = 1.52 × 10−9, Kruskal–Wallis test; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001; Steel–Dwass post hoc test). Data are represented as means ± SDs.
(C) TEM images of the cerebral cortex of marmosets. Ultrathin sections were prepared from brain slices cleared with ScaleSF (C1–C3) or stored in PBS(−) (C4–C6). Marmoset brains were fixed with 4% PFA (C1 and C4), 4% PFA containing 0.2% (C2 and C5), or 1% GA (C3 and C6) (n = 4 marmosets). Brain slices were cleared with ScaleSF and embedded in ScaleS4 gel for 24 hr, or stored in PBS(−) at 4°C in the analysis. Arrowheads indicate postsynaptic membranes. Scale bar, 500 nm.
See also Figures S4 and S5.