Abstract
It is important to understand changes in cell distribution that occur as a part of disease progression. This is typically achieved using standard sectioning and immunostaining, however, many structures and cell distribution patterns are not readily appreciated in two-dimensions, including the distribution of neural stem and progenitor cells in the mouse forebrain. Three-dimensional immunostaining in the mouse brain has been hampered by poor penetration. For this reason, we have developed a method that allows for entire hemispheres of the mouse brain to be stained using commercially available antibodies. Brains stained for glial fibrillary acidic protein, doublecortin and nestin were imaged in three-dimensions using optical projection tomography and serial two-photon tomography. This staining method is simple, using a combination of heat, time and specimen preparation procedures readily available, so that it can be easily implemented without the need for specialized equipment, making it accessible to most laboratories.
Highlights
The spatial distribution of various cell types or proteins is fundamental to understanding normal and pathological processes in the brain
We found that increasing the temperature to 37uC using a histology microwave increased the dextran FITC penetration and produced a fluorescence intensity profile more consistent with free diffusion
Decreasing the concentration of the fixative used from 4% paraformaldehyde (PFA) to 1% PFA modestly increased the depth of penetration further
Summary
The spatial distribution of various cell types or proteins is fundamental to understanding normal and pathological processes in the brain. Comparison between control and experimental groups in a study routinely requires cutting and identification of equivalent sections in multiple specimens, a subjective process that can be difficult even in simple cases For these and other reasons, several optical imaging methods have been developed that enable imaging of the mouse brain directly in three-dimensions (3D) [1,2,3,4]. Examples include optical projection tomography (OPT) [5,6], light sheet fluorescent microscopy [7,8,9], blockface imaging [10,11], and serial twophoton tomography [4] With many of these tools, cell types or gene products of critical interest can be visualized using transgenic optical markers, such as fluorescent proteins, under the control of appropriate promoters. The appropriate transgenic mouse is not always available and it is impractical and expensive to generate such mice for studies where multiple markers are necessary simultaneously or where the breeding is already complicated due to the disease model being investigated
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