Immunohistochemistry is a widely used technique for detecting the presence, location, and relative abundance of antigens in situ. This introductory level protocol describes the reagents, equipment, and techniques required to complete immunohistochemical staining of rodent brain tissue, using markers for microglia and neuronal elements as an example. Specifically, this paper is a step-by-step protocol for fluorescent visualization of microglia and neurons via immunohistochemistry for Iba1 and Pan-neuronal, respectively. Fluorescence double-labeling is particularly useful for the localization of multiple proteins within the same sample, providing the opportunity to accurately observe interactions between cell types, receptors, ligands, and/or the extracellular matrix in relation to one another as well as protein co-localization within a single cell. Unlike other visualization techniques, fluorescence immunohistochemistry staining intensity may decrease in the weeks to months following staining, unless appropriate precautions are taken. Despite this limitation, in many applications fluorescence double-labeling is preferred over alternatives such as 3,3'-diaminobenzidine tetrahydrochloride (DAB) or alkaline phosphatase (AP), as fluorescence is more time efficient and allows for more precise differentiation between two or more markers. The discussion includes troubleshooting tips and advice to promote success.

Item Type:	Contribution to Refereed Journal
Keywords:	neuroscience, immunohistochemistry, Iba1, pan-neuronal, fluorescence, double-labeling, microglia, neuron, rat, antibody
Research Division:	Medical and Health Sciences
Research Group:	Neurosciences
Research Field:	Neurosciences not elsewhere classified
Objective Division:	Health
Objective Group:	Clinical Health (Organs, Diseases and Abnormal Conditions)
Objective Field:	Nervous System and Disorders
UTAS Author:	Ziebell, JM (Dr Jenna Ziebell)
ID Code:	101716
Year Published:	2015
Web of Science® Times Cited:	7
Deposited By:	Wicking Dementia Research and Education Centre
Deposited On:	2015-07-02
Last Modified:	2016-11-14
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