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

Summary

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

Abstract

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.

Introduction

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.

Protocol

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 hazardous chemicals (like formalin, AEC and xylene), should be followed (e.g., fume hoods).

1. Formalin fixation of tissues

1. Place 3-5 mm brain tissues collected after necropsy¹³ 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 to processing, place the tissue in 70% ethanol.

2. Tissue processing

1. Following the specimen fixation, dissect the tissue to include the important brain areas e.g., cross sections of the brainstem, cerebellum (3 lobes), or the hippocampus (both hippocampi) each cut 3 to 5 mm thick and placed into processing cassettes.
2. Process the tissue cassettes for paraffin wax infiltration, embedded into paraffin blocks and sectioned (3 to 6 µm) on a microtome.

3. Preparation of Materials / Staining dishes

1. Set up the staining dish¹⁴ (Table of Materials) as shown in Figure 1. Fill each dish with 250 mL of the solution.
2. Preparation of the 3-Amino-9-ethylcarbizole (AEC) substrate stock solution
   1. Dissolve one 20 mg tablet of 3-amino 9-ethylcarbazole (AEC) into 5 mL of N,N, dimethylformamide using a glass pipette.
      CAUTION: AEC is a carcinogen.
3. Preparation of the protease (e.g., Pronase) stock solution for the antigen retrieval
   1. Dissolve 7 mg of the protease in 200 mL of PBS.
4. Preparation of the rinse buffer PBS-T
   1. Add 10 mL of Tween 80 to 990 mL of PBS. Mix it well to form a homogenous solution.

4. Deparaffinization and tissue rehydration

1. Make 5 µm paraffin section using a microtome, float it on a water bath at 38 ˚C and collect on to glass slides. Label the slides with a reagent resistant pen/marker.
2. Place the slides onto a tray and melt in a 55-60 ˚C oven for 1 h. Do not raise the temperature above 60 ˚C as it can destroy the viral antigen.
3. Remove slides from the oven and immediately deparaffinize in 3 consecutive xylene rinses of 5 min each in the dishes 1, 2 and 3.
4. Rehydrate the sections on the slide by sequential immersions in decreasing dilution of ethanol to deionized water: (4 through 11 is a dip rinse) dish 4: xylene/100% ethanol (1:1); dish 5: 100% ethanol; dish 6: 100% ethanol; dish 7: 95% ethanol; dish 8: 95% ethanol; dish 9: 80% ethanol; dish 10: 70% ethanol; dish 11: deionized water (Figure 1. Dish set-up).
   CAUTION: Xylene is a hazardous chemical and work should be conducted in a fume hood.

5. Proteolytic antigen retrieval

1. Treat slides with the protease (2.5 µg/mL of PBS) for 30 min for proteolytic antigen retrieval in dish 12.
2. Then rinse in PBS-T for 10 min (dish 13).
3. Treat with 3% hydrogen peroxide for 10 min (dish 14).
4. Again, wash with PBS-T for 10 min (dish 15).

6. Staining procedure

1. Handle slides one at a time keeping the remaining slides submerged in the buffer (do not remove the whole slide holder - keep slides wet). Remove one slide and blot off excess buffer (using a paper towel) from around the tissue section being careful not to disturb the tissue section. Incubate slides in a humidity chamber, made by placing moistened paper towels, on the lab bench top¹⁴ at room temperature with normal goat serum (blocking) for 15 min.
2. Incubate with the optimal pre-determined dilution primary anti-rabies antibody (1:250 dilution of mouse anti-rabies serum, unpublished) (positive control) and negative control antibodies at room temperature same as above (step 6.1.) for 60 min with no washes in between.
3. After 60 min, wash with PBS-T for 10 min (dish 16).
4. Incubate with the biotinylated antibody (species specific) in a humidity chamber at room temperature for 15 min (handling same as step 6.1.).
5. Wash with PBS-T for 10 min (dish 16).
6. Incubate with Streptavidin-HRP complex in a humidity chamber at room temperature for 15 min (handling same as 6.1.).
7. Wash with PBS-T for 10 min (dish 16).
8. Incubate with peroxidase substrate, amino-ethylcarbazole (AEC), in a humidity chamber at room temperature for 10 min. Make AEC just prior to use. To do so, add 1 mL of AEC stock solution to 14 mL of 0.1M acetate buffer, pH 5.2. Add 0.15 mL of 3% H₂O₂. Filter the mixture just before use (0.45 µm filter).
   NOTE: The working solution of AEC is only stable for 2-3 h. The AEC stock solution can be stored in the refrigerator for longer periods.
9. Wash in deionized water 10 min (dish 17)
10. Counterstained with Gill's Hematoxylin diluted 1:2 with deionized water for 2 min (dish 18).
11. Rinse off excess Hematoxylin with deionized water dip rinse (dish 19 and 20).
12. Rinse in Scott's Tap water 30 s (bluing solution) dish 21.
13. Wash in deionized water 10 min (dish 22)
14. Remove slides one at a time - mount with water-soluble mounting medium.
15. Read slides on a light microscope.

Representative Results

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 2D-F correspond to the cerebellum and Purkinje cells; and Figure 2G-I correspond to the hippocampus. Figure 2C,F,I are negative control samples. The magenta red staining demonstrates the color development using AEC substrate in the blue background (Hematoxylin counterstain) due to the reactivity of antibodies against rabies antigen. AEC is a peroxidase substrate, which upon oxidation reaction, catalyzed by HRP, results in water insoluble precipitate observable under a light microscope.

A positive result in IHC corresponds to magenta red staining in tissue sections. The staining of cytoplasmic inclusions and granular inclusions of varying size are indicative of samples positive for RABV infections. Samples are deemed negative if no specific red staining or only the blue background due to hematoxylin was observed. In addition to the positive staining, the distribution of inclusions could provide indirect quantification of levels of rabies antigen in the sample, which might correspond to the viral load of the tissue samples. Irrespective of the levels of distribution, any specific staining will classify the sample as positive for RABV antigen detection.

Figure 1: Flow chart indicating different steps for IHC testing. Please click here to view a larger version of this figure.

Figure 2: Immunohistochemical staining of positive and negative rabies brain tissue. (A) Intracytoplasmic viral inclusions and rabies virus antigen detection in the brainstem 200x total magnification; (B) positive brainstem 400x; (C) brainstem negative control 200x; (D) rabies virus inclusions within the cerebellum 200x; (E) cerebellum and Purkinje cells 400x; (F) cerebellum negative control; (G) Viral inclusions within hippocampus 200x; (H) hippocampus 400x; hippocampus negative control 200x. The red stain indicates the presence of rabies virus antigen using the Streptavidin-biotin complex staining method (AEC substrate). Hematoxylin counterstain (blue). Please click here to view a larger version of this figure.

Discussion

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 of side chains like cross-linking, the samples need to be processed before the antigen detection. In this protocol, the epitopes were recovered through the partial proteolytic digestion by the protease (e.g., Pronase) to enable the binding of primary antibodies to the RNP complex. While mAbs reactive against N protein are predominantly relied on in DFA and DRIT, pAbs that are reactive against multiple epitopes on N protein would be preferred for an IHC test. In addition, the reactivity or pAbs could be broader against different RABV variants and against non-rabies lyssaviruses as compared to mAbs.

One of the major limitations of IHC test is the protocol involves several sequential steps and takes about 6 hours for completion. If the tissue needs to be fixed in formalin and embedded in paraffin blocks, it requires an additional 1 - 2 days before the tissue could be stained. Another limitation is the non-availability of commercial primary anti-rabies antibodies for the IHC test. However, IHC does provide an option to perform rabies diagnosis when only FF tissues are available for testing. The IHC test is particularly important for testing rabies cases if one half of the tissues are stored in formalin (and other unfixed tissues tested by DFA) and it was necessary to test complete cross section of the brainstem and other tissues, as required for diagnosis. Rabies antigen detection by IHC test can be utilized for human post-mortem brain samples for the diagnosis and / or retrospective analysis of suspect cases based on the clinical symptoms. While IHC was not approved as a primary or confirmatory test for rabies diagnosis, like DFA, the method detects antigen using rabies specific antibodies. Comparison of DFA using fresh/frozen vs FF tissues provided similar sensitivity and specificity¹⁵. Unless antibodies are directly conjugated to FITC (the requirement for DFA test), HRP labeled antibodies can be used in IHC for staining rabies antigen. The advantage with HRP based detection is the ability to use a light microscope for the observation. The current commercially available DFA reagents, FITC conjugated rabies specific antibodies (mAbs) does not detect antigen after formalin fixation due to the modification of epitopes. However, if FITC conjugated rabies specific pAbs are available, it can be used as a staining method, as recommended by World Health Organization¹⁶. In addition to antigen detection, FF tissues can be subjected to RNA isolation followed by PCR and sequencing using specific primers for confirming the presence of RABV genomic RNA.

The other advantages of formalin-fixed tissues include determination of histological changes by hematoxylin and eosin staining method. While the formalin treatment preserves protein, it completely inactivates most pathogens in the sample due to the extensive crosslinking of proteins and degradation or modification of nucleic acids. Thus, the method improves the safety of biological sample handling, shipping and testing compared to DFA. The acetone fixation step in DFA does not inactivate RABV and should be handled with appropriate PPE¹⁷. The samples after formalin fixation are stable and can be stored at ambient temperature, which is suited for low-resource areas where access to a cold storage is limited. Similarly, paraffin-embedded formalin-fixed tissues can be considered for the long-term storage at ambient temperatures without losing the antibody reactivity against proteins.

Materials

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