CLARITY increases sensitivity and specificity of fluorescence immunostaining in long-term archived human brain tissue

Abstract

AbstractImmunohistochemistry on archival human brains is often limited because several conditions arise that complicate the use for high-resolution fluorescence microscopy. In this study, we developed a novel clearing approach for immunofluorescence-based analysis of perfusion- and immersion-fixed post mortem human brain tissue, termed hCLARITY. hCLARITY is optimized for specificity by reducing off-target labeling and yields very sensitive stainings in human brain sections allowing for super-resolution microscopy with unprecedented imaging of pre- and postsynaptic compartments. Moreover, hallmarks of the Alzheimer’s disease were preserved with hCLARITY, and importantly classical DAB or Nissl stainings are compatible with this protocol. hCLARITY is extremely versatile as demonstrated by the use of more than 30 well performing antibodies and allows for de- and subsequent re-staining of the same tissue section, which is important for multi-labelling approaches, e.g., in super-resolution microscopy. Taken together, hCLARITY enables research of the human brain with highest sensitivity and down to sub-diffraction resolution and therefore has enormous potential for the investigation of local morphological changes, e.g., in neurodegenerative diseases.Striking Image / Graphical abstractSummary of the main advantages of hCLARITY. CLARITY was applied on human post mortem brain tissue in direct comparison to untreated sections (minus CLARITY). Owing to removal of lipids during the clearing with SDS and the resulting reduction in light scattering, cleared sections were less opaque. The denaturing effect of the detergent SDS most likely results in better accessibility of certain epitopes. The major benefits of hCLARITY were found in an increased sensitivity and specificity of antibodies for neuronal cells. The adapted hCLARITY protocol is compatible with staining techniques for confocal microscopy, super-resolution microscopy, and light microscopy.