Optical Clearing and Immunolabeling of Archival Human Prenatal Neural Tissue Blocks

Traditional histological approaches using tissue sections of conventional thickness (7–100 μm) fail to fully capture the intricate three-dimensional architecture of neuronal and glial cells. Visualization of larger tissue blocks is hindered by tissue opacity and limited antibody penetration, where opacity arises from light scattering at interfaces with mismatched refractive indices (RIs). Objective: Сompare two RI-matching methods, CLARITY and SWITCH, for processing archival human fetal brain tissue blocks fixed long-term in 4% buffered paraformaldehyde at room temperature. Results: Cerebral cortex blocks from four human fetuses (25–39 gestational weeks) were examined. Confocal microscopy revealed imaging depths of up to 2 mm for both methods. However, CLARITY exhibited shallow antibody penetration, with staining predominantly restricted to superficial layers (150– 180 μm), while SWITCH enabled uniform antibody penetration beyond 300 μm. Our findings demonstrate that both CLARITY and SWITCH are applicable to archival human fetal brain tissue with prolonged fixation (up to 3.5 years). Conclusion: SWITCH outperformed CLARITY in tissue processing time, antibody penetration depth, and low post-treatment sample deformation.