We develop protocols for advanced immunohistochemistry in fixed brain tissue to facilitate two-dimensional and three-dimensional imaging of molecular patterns to reveal defined cellular and histological structures. We work primarily with mouse tissue, but have also developed adaptations of our approach for rat, marmoset, and human brain tissues. Our methods can be divided into two inter-related approaches: thick-section IHC and whole-mount IHC.
Both methods leverage delipidation and tissue clearing technology developed by our group to render tissues transparent to light across the wavelengths used in fluorescent microscopy. By doing so, we are able to capture patterns and structures across a larger depth of tissue.
In thick-section IHC, we perform immunolabeling on 100-250μm sections of delipidated brain tissue, employ high refractive-index mounting media to optically clear the thick section when mounted on slides, and then image our section in 2D or 3D using widefield fluorescent microscopy. This approach is analogous to classical histological approaches, and the results obtained are consistent with those of traditional histology.
In whole-mount IHC, we perform immunolabeling on delipidated whole brains or brain hemispheres, optically clear the intact tissue using the iDISCO-family methods, and then image the entire brain or hemisphere in 3D using lightsheet microscopy. This method allows us to perform unbiased surveys of cell populations in the brain and/or of proteomic changes that occur in the brain in both health and disease.
Thick-section IHC can be employed as a standalone approach to biological inquiry, or as a precursor step to whole-mount IHC. Below, we provide our up-to-date optimized protocols for tissue collection, delipidation, and thick-section IHC, as well as a link to our previously published iDISCO+ whole mount method. We hope these resources will provide accessible guidance to labs hoping to try out these approaches for the first time, as well as serve useful resources and references to scientists previously experienced with these methods.