Free-floating Immunostaining of Mouse Brains

Authors

Longlong Tu
Nan Zhang
Kristine M Conde
Jonathan C Bean
Chunmei Wang
Yong Xu

Publication Date

10-7-2021

Journal

Journal of Visualized Experiments

DOI

10.3791/62876

PMID

34694284

PMCID

PMC8981791

PubMedCentral® Posted Date

4-5-2022

PubMedCentral® Full Text Version

Author MSS

Published Open-Access

yes

Keywords

Animals, Brain, Extracellular Matrix, Mice, Reproducibility of Results, Staining and Labeling

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 intra-aortic 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.