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.
All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.
1. Formalin fixation of tissues
2. Tissue processing
3. Preparation of Materials / Staining dishes
4. Deparaffinization and tissue rehydration
5. Proteolytic antigen retrieval
6. Staining procedure
Figure 1: Flow chart indicating different steps for IHC testing.
The authors have nothing to disclose.
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 |