Protocol References
Here you can access up-to-date versions of our thick-section immunohistochemistry protocols. If you have a question about these protocols which is not answered on this or the FAQ Page, please do not hesitate to reach out to us (see Contact Us).
Overview
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.
Reference Modules
01.
Tissue Collection
This is our optimized mouse brain tissue collection protocol. We dissect the brain immediately after transcardial perfusion. Perfusion preserves tissue structure via fixation and minimizes vasculature background by off-target immunolabeling. A successful perfusion is important for obtaining clean section staining results and is essential for producing usable whole-mount results.
02.
Sample Delipidation
This delipidation protocol is used on tissue samples intended for either thick-section or whole-mount IHC. Removing lipids from fixed tissue minimizes light scattering off of membranous structures and leaves behind a protein scaffold which can later be homogenized through use of a refractive-index (RI) matched media. Delipidation is achieved through a combination of hydrophilic and hydrophobic solvent washes using commonly found, inexpensive laboratory chemicals.
03.
Thick-Section Immunohistochemistry
This IHC protocol is used to validate antibodies on thick-sections for compatibility with iDISCO whole-mount immunolabeling. For researchers who do not have access to a lightsheet microscope or for whom whole-mount approaches are not essential, using this easy thick-section method can allow the researcher to examine localized protein targets in three-dimensions in specific known brain areas of interest. Unlike traditional thin-section IHC, this thick-section protocol leverages delipidated brain tissue to enable imaging over a 100-250μm depth. Therefore, it can also be used to rapidly screen through the entire brain regions with parallel processing of a few dozen serial sections. Beyond increasing imaging depth, using delipidated tissue for section staining provides greater access to lipid-enriched regions which facilitates complete penetration and staining of white-matter structures like myelinated axon tracts.
01.
Tissue Collection
This is our optimized mouse brain tissue collection protocol. We dissect the brain immediately after transcardial perfusion. Perfusion preserves tissue structure via fixation and minimizes vasculature background by off-target immunolabeling. A successful perfusion is important for obtaining clean section staining results and is essential for producing usable whole-mount results.
02.
Sample Delipidation
This delipidation protocol is used on tissue samples intended for either thick-section or whole-mount IHC. Removing lipids from fixed tissue minimizes light scattering off of membranous structures and leaves behind a protein scaffold which can later be homogenized through use of a refractive-index (RI) matched media. Delipidation is achieved through a combination of hydrophilic and hydrophobic solvent washes using commonly found, inexpensive laboratory chemicals.
03.
Thick-Section Immunohistochemistry
This IHC protocol is used to validate antibodies on thick-sections for compatibility with iDISCO whole-mount immunolabeling. For researchers who do not have access to a lightsheet microscope or for whom whole-mount approaches are not essential, using this easy thick-section method can allow the researcher to examine localized protein targets in three-dimensions in specific known brain areas of interest. Unlike traditional thin-section IHC, this thick-section protocol leverages delipidated brain tissue to enable imaging over a 100-250μm depth. Therefore, it can also be used to rapidly screen through the entire brain regions with parallel processing of a few dozen serial sections. Beyond increasing imaging depth, using delipidated tissue for section staining provides greater access to lipid-enriched regions which facilitates complete penetration and staining of white-matter structures like myelinated axon tracts.