University Of Tasmania

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Primer for immunohistochemistry on cryosectioned rat brain tissue: example staining for microglia and neurons

journal contribution
posted on 2023-05-22, 02:33 authored by Evilisizor, MN, Ray-Jones, HF, Lifshitz, J, Jenna ZiebellJenna Ziebell
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.


Publication title

Journal of visualized experiments : JoVE





Article number







Wicking Dementia Research Education Centre


MYJoVE Corporation

Place of publication

United States

Rights statement

Copyright 2015 Journal of Visualized Experiments

Repository Status

  • Restricted

Socio-economic Objectives

Clinical health not elsewhere classified