Name: immunohistochemistry test for the lyssavirus antigen detection from formalin-fixed tissues
Uploaded: 2021-10-26T13:00:00
Description: Watch this Scientific Journal Video about Immunohistochemistry Test for the Lyssavirus Antigen Detection from Formalin-Fixed Tissues at JoVE.com

We thank the laboratorians, epidemiologists, and affiliates with public health departments for sample submissions to the Centers for Disease Control and Prevention. The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention. Use of trade names and commercial sources are for identification only and do not imply endorsement by the Centers for Disease Control and Prevention.

While the IHC protocol was performed on formalin-fixed tissues, which inactivates RABV if present, appropriate biosafety protocols should be properly followed. All biosafety procedures are described in the Biosafety in Microbiological and Biomedical Laboratories (BMBL) 5th Edition (https://www.cdc.gov/biosafety/publications/bmbl5/index.htm), including wearing proper personal protective equipment (PPE), and vaccination requirement as described12. In addition, proper containment and handling of haza...

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Due to the high fatality rate of rabies after the symptom onset, the diagnosis of suspect animals for RABV infection is extremely critical for an appropriate post-exposure prophylactic treatment. Rabies diagnosis primarily depends on DFA, DRIT, and PCR-based techniques using fresh or frozen tissues. For testing of formalin-fixed tissues, the IHC test provides an alternative method for the sensitive and specific detection of RABV antigen. While the tissues fixed in formalin have proteins stabilized due to the modification...

Rabies is an acute progressive encephalitis caused by the negative sense RNA viruses belonging to the genus lyssavirus1. Nearly 99% of all human deaths caused by the infection with rabies virus (RABV), the type member of the genus, is transmitted by dogs2. Rabies diagnosis of suspect animals relies on the detection of antigen (primarily viral encoded nucleoprotein, N protein) in complex with genomic RNA (ribonucleoprotein complex, RNP) in the brain tissue3. The antigen detection by the direct fluorescent antibody (DFA) test is considered the gold standard for rabies diagnosis4. The method utilizes fresh or fresh frozen brain material, a touch impression on a slide, fixation in acetone, staining using commercially available fluorescent isothiocyanate (FITC) labeled monoclonal or polyclonal antibodies (mAbs/pAbs) and read by the fluorescence microscopy5. The DFA test is rapid, sensitive, and specific for rabies antigen detection in fresh brain tissue. Recently, a direct rapid immunohistochemical test (DRIT), modified immunohistochemistry (IHC) technique, was demonstrated to exhibit similar sensitivity to DFA but offers the advantage of light microscopy for visualization6. While the detection method used in DRIT, is similar to IHC, the initial step utilizes fresh or frozen tissues to generate touch impressions of the sample followed by fixation in formalin.
IHC is a widely used technique to determine histological changes and detection of proteins using specific antibodies in formalin-fixed tissues embedded in paraffin blocks. IHC is an established alternative test for the rabies antigen detection in the tissue sections7. IHC has been particularly utilized for the diagnosis of retrospective cases that exhibited neurological diseases to determine the burden of rabies8. Paraffin-embedded formalin-fixed tissues preserve the proteins for the detection even after several years when stored at ambient temperature9. Formalin treatment modifies proteins by cross-linking and altering the amino acid side chains, which might make the epitopes no longer reactive against antibodies10. While the IHC test for rabies antigen detection involves either mAbs or pAbs, the latter is advantageous as multiple epitopes and divergent lyssaviruses can be detected11.
The standard steps involved in IHC are formalin fixation of tissues, embedding in paraffin blocks, sectioning of tissues, deparaffinization and hydration, epitope recovery, reactivity against primary and secondary antibodies, and the development using chromogenic substrates. This manuscript describes a detailed account of the protocol for rabies diagnosis. For rabies antigen detection, mouse serum immunized with RABV (pAbs) generated at the U.S. Centers for Disease Control and Prevention (CDC) Atlanta, Georgia, in combination with biotinylated anti-mouse secondary antibodies are utilized. Biotinylated Abs are detected by the addition of streptavidin-horseradish peroxidase (HRP) complex followed by the color development with amino-ethylcarbazole substrate.

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			<td>3% hydrogen peroxide</td>
			<td>Pharamacy brands</td>
			<td></td>
			<td>Off the shelf 3% H2O2</td>
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			<td>3-Amino-9-ethylcarbazole (AEC)</td>
			<td>Millipore Sigma</td>
			<td>A6926</td>
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			<td>Acetate Buffer pH 5.2</td>
			<td>Poly Scientific R&#38;D Corp.</td>
			<td>s140</td>
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			<td>Buffered Formalin 10% Phosphate Buffered</td>
			<td>Fisher Scientific</td>
			<td>SF100-4</td>
			<td>Certified</td>
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			<td>Cover slips Corning</td>
			<td>Fisher Scientific</td>
			<td>12-553-471</td>
			<td>24 X 50 mm</td>
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			<td>Ethanol 190 Proof</td>
			<td>Pharmco-AAPER</td>
			<td>111000190</td>
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			<td>Ethanol 200 Proof</td>
			<td>Pharmco-AAPER</td>
			<td>111000200</td>
			<td></td>
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			<td>Gill&#39;s hematoxylin formulation #2</td>
			<td>Fisher Scientific</td>
			<td>CS401-1D</td>
			<td></td>
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			<td>HistoMark Biotin-Streptavidin Peroxidase Kit</td>
			<td>seracare</td>
			<td>71-00-18</td>
			<td>Mouse Primary Antibody&#160;</td>
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			<td>ImmunoHistoMount</td>
			<td>Millipore Sigma</td>
			<td>i1161</td>
			<td>Mounting media</td>
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			<td>N,N, Dimethyl formamide GR</td>
			<td>Fisher Scientific</td>
			<td>D119</td>
			<td></td>
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			<td>Phosphate Buffered Saline&#160;</td>
			<td>HyClone</td>
			<td>RR14440.01</td>
			<td>01M, pH 7.2 (pH 7.2-7.6)</td>
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			<td>Plan-APOCHROMAT 40X/0.95 Objective</td>
			<td>Multiple vendors</td>
			<td></td>
			<td></td>
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			<td>Plan-APOCHROMATIC 20X/0.75 Objective</td>
			<td>Multiple vendors</td>
			<td></td>
			<td></td>
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			<td>Pronase</td>
			<td>Millipore Sigma</td>
			<td>53702</td>
			<td>Protease, Streptomyces griseus</td>
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			<td>Scott&#39;s Tap Water&#160;</td>
			<td>Poly Scientific R&#38;D Corp.</td>
			<td>s1887</td>
			<td></td>
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			<td>Tissue-Tek Slide stain set</td>
			<td>Fisher Scientific</td>
			<td>50-294-72</td>
			<td></td>
		</tr>
		<tr>
			<td>TWEEN-80&#160;</td>
			<td>Millipore Sigma</td>
			<td>P1754</td>
			<td></td>
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			<td>Xylene</td>
			<td>Fisher Scientific</td>
			<td>X3S-4</td>
			<td>Histological Grade</td>
		</tr>
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			<td>Zeiss Axioplan 2 imaging - microscope</td>
			<td>Multiple vendors</td>
			<td></td>
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immunohistochemistry test for the lyssavirus antigen detection from formalin-fixed tissues

One of the primary diagnostic modalities for rabies is the detection of viral ribonucleoprotein (RNP) complex (antigen) in the infected tissue samples. While the direct fluorescent antibody (DFA) test or the direct rapid immunohistochemical test (DRIT) are most commonly utilized for the antigen detection, both tests require fresh and/or frozen tissues for impressions on slides prior to the antigen detection using antibodies. If samples are collected and fixed in formalin, neither test is optimal for the antigen detection, however, testing can be performed by conventional immunohistochemistry (IHC) after embedding in paraffin blocks and sectioning. With this IHC method, tissues are stained with anti-rabies antibodies, sections are deparaffinized, antigen retrieved by partial proteolysis or other methods, and incubated with primary and secondary antibodies. Antigens are stained using horseradish peroxidase / amino ethyl carbazole and counterstained with hematoxylin for the visualization using a light microscope. In addition to the specific antigen detection, formalin fixation offers other advantages like the determination of histological changes, relaxed conditions for specimen storage and transport (under ambient temperatures), ability to test retrospective cases and improved biological safety through the inactivation of infectious agents.

Figure 2 demonstrates representative IHC staining results of positive and negative control samples in different brain tissues tested. Figure 2A,D,G represent positive samples at 200x, while Figure 2B,E,H correspond to 400x magnification, respectively. Figure 2A-C correspond to the brainstem; Figure 2...

Watch this Scientific Journal Video about Immunohistochemistry Test for the Lyssavirus Antigen Detection from Formalin-Fixed Tissues at JoVE.com

Immunohistochemistry Test for the Lyssavirus Antigen Detection from Formalin-Fixed Tissues

Here, we present an immunohistochemistry test protocol for the detection of rabies virus antigen as an alternative diagnostic test for formalin-fixed tissues.

One of the primary diagnostic modalities for rabies is the detection of viral ribonucleoprotein (RNP) complex (antigen) in the infected tissue samples. ...

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