Free-floating immunostaining of mouse brains

Authors: TU, LONGLONG - Children'S Nutrition Research Center (CNRC); ZHANG, NAN - Children'S Nutrition Research Center (CNRC); CONDE, KRISTINE - Children'S Nutrition Research Center (CNRC); BEAN, JONATHAN - Children'S Nutrition Research Center (CNRC); WANG, CHUNMEI - Children'S Nutrition Research Center (CNRC); XU, YONG - Children'S Nutrition Research Center (CNRC)

Submitted to: The Journal of Visualized Experiments (JoVE)
Publication Type: Review Article
Publication Acceptance Date: 8/24/2021
Publication Date: 10/7/2021
Citation: Tu, L., Zhang, N., Conde, K., Bean, J., Wang, C., Xu, Y. 2021. Free-floating immunostaining of mouse brains. The Journal of Visualized Experiments (JoVE). 176. Article e62876. https://doi.org/10.3791/62876.
DOI: https://doi.org/10.3791/62876

Interpretive Summary:

Technical Abstract: Immunohistochemical staining of mouse brains is a routine technique commonly used in neuroscience to investigate central mechanisms underlying the regulation of energy metabolism and other neurobiological processes. However, the quality, reliability, and reproducibility of brain histology results may vary among laboratories. For each staining experiment, it is necessary to optimize the key procedures based on differences in species, tissues, targeted proteins, and the working conditions of the reagents. This paper demonstrates a reliable workflow in detail, including intra­aortic perfusion, brain sectioning, free-floating immunostaining, tissue mounting, and imaging, which can be followed easily by researchers in this field. Also discussed are how to modify these procedures to satisfy the individual needs of researchers. To illustrate the reliability and efficiency of this protocol, perineuronal nets were stained with biotin-labeled Wisteria florbunda agglutinin (WFA) and arginine vasopressin (AVP) with an anti-AVP antibody in the mouse brain. Finally, the critical details for the entire procedure have been addressed, and the advantages of this protocol compared to those of other protocols. Taken together, this paper presents an optimized protocol for free-floating immunostaining of mouse brain tissue. Following this protocol makes this process easier for both junior and senior scientists to improve the quality, reliability, and reproducibility of immunostaining studies.