FIGURE 1.

Sample processing. (A) Conceptual diagram of the sample processing pipeline. Human brain slices with a thickness greater than 100 μm were cleared with different clearing protocols (CLARITY, SWITCH, and SHIELD) and then immunolabeled (IF, immunofluorescence). Slices with a thickness less than 100 μm were stained before to perform ExM. All the samples were acquired with different optical techniques. Created with BioRender.com. (B) CLARITY sandwich preparation for hydrogel inclusion. Human brain slices were placed on two coverslips, separated by a 500 μm-thick stainless steel spacer. The sandwich was then filled with a hydrogel CLARITY solution using a syringe and placed in a 50-ml tube dipped with the same solution and incubated at 4°C. After the samples were degassed with nitrogen (N₂) removing the oxygen (O₂). The temperature was increased to 37°C to initiate polymerization. Finally, the embedded sample was extracted from the gel and washed with a clearing solution to remove lipids. Created with BioRender.com. (C–F) Representative images of human brain slices pre-clearing (in PBS) and after refractive index matching in 68% TDE: CLARITY (A), SWITCH (B), and SHIELD (C) and dH₂O (ExM) (F).