Researchers study neural tissues, neural cell markers and neural-specific protein targets to better understand the nervous system, a complex collection of highly specialized cells that are involved in signal transmission throughout the body. By using antibodies to identify neuronal cells via established techniques such as immunocytochemistry (ICC) or immunohistochemistry (IHC), it is possible to unravel signaling pathways, monitor cell growth and development, and to elucidate disease mechanisms. However, to achieve meaningful data from inherently fragile and often limited sample material requires highly characterized antibodies and experiments that are thoroughly optimized. MilliporeSigma scientists have shared with us their expertise in using ICC and IHC to study neuroscience targets.
Neurons, the principle cells of the nervous system, can be broadly categorized as sensory, motor, or interneurons; however, the distinction between different cell types is vastly more complex, especially within the brain. Classified by features such as morphology, polarity, and biochemical characteristics, and supported in their function by glial cells that include astrocytes, oligodendrocytes, and Schwann cells, neuronal diversity can make the study of these cells extremely challenging. Thorough research is key to understanding best practices for cell handling and treatment, and for ascertaining whether detection of the target antigen of interest is to be expected in a certain sample type. A literature review can also inform whether investigation of the chosen target is appropriate to address a specific hypothesis.
Researchers wishing to study neuroscience targets via ICC or IHC typically work with fixed monolayer cells, thin tissue sections, organoids, or thick samples of tissue such as brain. Although it is common practice to generate 2-dimensional images from plated cells or tissue sections, researchers are increasingly turning to 3D imaging to better capture features such as neuronal structure and dynamic orientation. For antibody reagents to access antigenic targets, cell monolayers and tissue sections may require permeation with a suitable detergent, while thicker samples often benefit from clearing with an agent such as Visikol® HISTO™.
High-quality antibody reagents are essential to any immunostaining experiment, and it is often recommended to select pre-validated antibodies with supporting data to confirm target-specificity. By studying product datasheets, researchers can easily identify antibodies that have been tested in the intended application and against a relevant form (native or denatured) of the target.
Image: IHC staining of rat cerebellum using anti-NeuN clone A60 from MilliporeSigma. Validated for applications which include ICC and IHC, this antibody has been referenced in >2700 publications to date.
Product datasheets often indicate an appropriate antibody dilution, although it is good practice to perform a titration when identifying an acceptable assay window for a specific sample type. This should of course be carried out using suitable controls, rather than wasting precious sample material. To reduce background staining, which can occur due to cross-reactivity, it may be prudent to choose monoclonal, recombinant, or cross-adsorbed antibodies.
MilliporeSigma offers antibodies that undergo both standard and enhanced validation, providing neuroscience researchers with confidence in their ICC and IHC data. Supplied with detailed datasheets illustrating tested applications and recommendations for staining success, and widely supported by publicly available data, these products are cited in thousands of publications including many renowned neuroscience journals.
Image: Immunofluorescence staining of astrocytes (green) in the rat cerebellum using anti-GFAP antibody, clone CL2713 from MilliporeSigma.
In addition to titrating antibody reagents, researchers should consider optimizing other key steps within their immunostaining protocol. For ICC and IHC these include fixation, permeabilization or clearing, blocking, antibody incubations, washing, and the choice of detection method. It is common practice to set up a checkerboard experiment to test multiple conditions in parallel while minimizing antibody use.
IHC staining of formalin-fixed paraffin-embedded (FFPE) tissue samples regularly necessitates an antigen retrieval step, since formalin crosslinks can mask antigens and prevent antibody binding. Product datasheets often indicate tried and tested antigen retrieval methods, and researchers may choose between heat-induced and proteolytic-induced epitope retrieval (HIER and PIER respectively).
When a fluorescent readout is chosen, the addition of mounting media can protect fluorophores from photobleaching and preserve the fluorescent signal. Many mounting medias also include a nuclear stain for convenience.
While the study of neuroscience is highly complex, ICC/IHC staining of neural targets needn’t be a chore. By thoroughly researching the target of interest, designing immunostaining protocols carefully, and using trusted antibody reagents, these powerful techniques can take your neuroscience research to the next level.