An Immunohistochemistry Staining for the Detection of Rabies Virus Antigens

Abstract

Source: Niezgoda, M. et al., Immunohistochemistry Test for the Lyssavirus Antigen Detection from Formalin-Fixed Tissues. J. Vis. Exp. (2021)

This video demonstrates immunohistochemistry staining for rabies virus antigen detection in infected mouse brain tissue. The process includes deparaffinization and alcohol treatment, followed by proteolytic treatment to enhance antigen accessibility. Immunohistochemistry staining reveals red precipitates, confirming the presence of rabies antigens in the tissue section.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Formalin fixation of tissues

1. Place 3-5 mm brain tissues collected after necropsy into 10% phosphate-buffered formalin solution (1:20 to 1:50 tissue/formalin ratio) for 24-72 h.
   CAUTION: Formalin is a toxic fixative.
2. Record the approximate tissue weight (size), tissue type (brain areas), and volume of formalin. It is important for laboratories to document and maintain records on fixation times.
3. For longer tissue storage after formalin fixation and prior t....

Results

Figure 1: Flow chart indicating different steps for IHC testing.

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Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
3% hydrogen peroxide	Pharamacy brands		Off the shelf 3% H2O2
3-Amino-9-ethylcarbazole (AEC)	Millipore Sigma	A6926
Acetate Buffer pH 5.2	Poly Scientific R&D Corp.	s140
Buffered Formalin 10% Phosphate Buffered	Fisher Scientific	SF100-4	Certified
Cover slips Corning	Fisher Scientific	12-553-471	24 X 50 mm
Ethanol 190 Proof	Pharmco-AAPER	111000190
Ethanol 200 Proof	Pharmco-AAPER	111000200
Gill's hematoxylin formulation #2	Fisher Scientific	CS401-1D
HistoMark Biotin-Streptavidin Peroxidase Kit	seracare	71-00-18	Mouse Primary Antibody
ImmunoHistoMount	Millipore Sigma	i1161	Mounting media
N,N, Dimethyl formamide GR	Fisher Scientific	D119
Phosphate Buffered Saline	HyClone	RR14440.01	01M, pH 7.2 (pH 7.2-7.6)
Plan-APOCHROMAT 40X/0.95 Objective	Multiple vendors
Plan-APOCHROMATIC 20X/0.75 Objective	Multiple vendors
Pronase	Millipore Sigma	53702	Protease, Streptomyces griseus
Scott's Tap Water	Poly Scientific R&D Corp.	s1887
Tissue-Tek Slide stain set	Fisher Scientific	50-294-72
TWEEN-80	Millipore Sigma	P1754
Xylene	Fisher Scientific	X3S-4	Histological Grade
Zeiss Axioplan 2 imaging - microscope	Multiple vendors