Fig. 2

Electron micrographs of human brain samples with different PMIs and cryo-substitution cocktails at X6500 magnification. a-d Frontal lobe from case 1 (PMI 16 h), a prepared with standard embedding method shows two myelinated axons; b After hybrid freezing with freeze substitution one, the myelinated axons have tightly wrapped myelin sheaths (arrows) with clearly visible microtubules inside the axons (arrow heads) and a strong overall contrast. c and d Samples prepared with substitution cocktail II (c) and cocktail III (d), exhibiting lower contrast and less preserved myelin sheath than (b). e-h Frontal lobe samples from case 4 (PMI 24 h), e prepared by standard embedding, f processed with cryo-substitution cocktail I (hybrid freezing), g hybrid freezing with cocktail II, and (h), hybrid freezing with cocktail III. f Shows the strongest contrast, but the myelin sheaths exhibit only a slight increase in preservation between (e) and (f) and no clear change in preservation in samples (g) and (h). Scale bars = 0.5 μm