Step-by-step protocol for chromogenic immunohistochemical staining of paraffin sections of animal organs in viral infections, using the example of SARS-CoV-2

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Introduction

In studies of the subcellular localization of viral proteins, immunohistochemical (IHC) staining is the method of choice [1]. Identifying the topography and distribution of viral particle protein structures in the tissues of model animals may indicate a direct or indirect influence of virions on the development of pathomorphological changes in organs. Such data are key to understanding viral pathogenesis and can facilitate diagnosis, prevention, and treatment of infection.

IHC analysis is based on the principle of conjugation between antibodies and antigens. There are two qualitative methods of IHC analysis: direct and indirect. The indirect method is considered more accurate, as it reduces the proportion of background staining [2]. Immunofluorescence staining is widely used in virology, both when working with cell cultures and when examining animal organ sections. Sections are prepared using the paraffin method or cryopreservation; both approaches allow for the preservation of viral antigens [3].

The IHC staining method has both advantages and disadvantages, particularly when working with viral markers in tissues. Viral proteins and the viral genome are distributed throughout the bloodstream within a few days of infection [4]. Consequently, the task of localizing clusters of viral structures becomes labor-intensive. Undoubtedly, the immunostaining technique involves a number of details that require special attention when working with certain structures.

The aim of this study is to develop an immunohistochemical staining protocol optimized for the detection of viral antigens in lung tissue using SARS-CoV-2 as an example.

Materials and methods

Viruses

The study was conducted using SARS-CoV-2 Omicron and Delta strains provided by staff members of the “Microorganism Collection” Department at the State Research Center “Vector”.

Laboratory animals

The study involved 6 female ferrets (Mustela putorius furo) aged 1 year with a body weight of 0.7–1.2 kg. The animals were housed in the vivarium of the State Research Center “Vector” at a relative humidity of 20% and a temperature of 15–24°C. The ferrets were housed in separate cages with visual and auditory contact maintained; under natural light/dark cycles, the animals had free access to food and water.

Work with the experimental animals was performed by staff of the Department of Zoonotic Infections and Influenza at the State Research Center “Vector.” Each stage of work with the animals was carried out in compliance with Recommendation No. 33 of the EEC Board dated November 14, 2023, “On Guidelines for Working with Laboratory (Experimental) Animals in Preclinical (Nonclinical) Studies.”¹. The study protocol was approved by the Bioethics Committee of the State Research Center “Vector” (Protocol No. 1 dated February 26, 2025).

Premedication of ferrets prior to euthanasia was performed by inducing general anesthesia with the combination anesthetic Zoletil 100 and the muscle relaxant Xyla. Each drug was administered intramuscularly at a dose of 3 mg/kg. Euthanasia was then performed using an automated compact CO₂ system (Euthanizer) to humanely remove the laboratory animals from the experiment. The CO₂ concentration (30% in stage 1, 70% in stage 2) and gas flow rate comply with the requirements of the American Veterinary Medical Association 2020².

Preparation of histological slides

After euthanasia and dissection of the animal’s lung tissue, the organs were fixed in formalin in accordance with the protocol in Appendix 1, Section 5 of “Methodological Guidelines 1.3.3103-13. Organization of the work of laboratories using electron and atomic force microscopy methods in the study of microbial cultures of pathogenicity groups I–IV."³. The transfer of tissue samples was conducted in accordance with biosafety requirements and the transfer protocols for inactivated preparations of Group 2 pathogenic microorganisms for light and electron microscopy No. 240125 dated February 3, 2025, and No. 070624 dated June 7, 2024, under the supervision of the Biosafety Department of the State Research Center “Vector.”

Animal lung samples were cut into pieces approximately 5 mm³ in size and then placed in histology cassettes. For tissue fixation and dehydration, an automatic vacuum processor Tissue-Tek VIP 6 AI (Sakura Finetek) was used, in which the samples were passed through a series of increasing alcohol concentrations (70, 80, 96, and 100%) followed by fixation in a xylene and ethanol solution. The cassettes were then immersed in a mixture of paraffin and xylene, where the samples were heated on the Tissue-Tek TEC 6 (Sakura Finetek) device panel for 2 hours to a constant temperature of 55°C. Paraffin blocks were formed at 5°C on the Tissue-Tek TEC (Sakura Finetek) instrument panel.

Next, thin sections of paraffin-embedded specimens were prepared using a Tissue-Tek AutoSection (Sakura Finetek) instrument. Preliminary experimental work revealed that for IHC staining, the optimal section thickness is no more than 2 μm, in contrast to routine histological studies, for which the section thickness can reach 5 μm [5]. Using a brush, a series of 3 sections was transferred to a water bath heated to 45°C. Next, the series of sections was mounted on slides with an adhesive coating. The slides were heated and dried in a TS-1/80 thermostat (Smolenskoye SKTB SPU) for 1 day at 55°C.

Several types of adhesive-coated slides were tested in this study. The surface adhesive coating enhances the adhesion and stable fixation of paraffin sections to the glass, which is particularly necessary during the high-temperature epitope retrieval stage in a buffer with a high pH value, such as Tris-EDTA [6]. Charged slides have a positively charged coating and are suitable for use with frozen or formalin-fixed paraffin sections. Slides coated with silane or poly-L-lysine have also demonstrated a high degree of adhesion with paraffin sections of animal organs, even under conditions of aggressive chemical environments.

Deparaffinization and epitope retrieval

In this study, deparaffinization and retrieval were performed using the Dartmon AS330 immunostainer (Dartmon, China). For the analysis of samples containing viral determinants, standard deparaffinization and retrieval protocols recommended by the manufacturer were used. Before starting the procedure, the slides were placed in the instrument and covered with a slide cover to create a humidity chamber effect (Fig. 1).

 

Fig. 1. Layout of the slide and protective device in the Dartmon AS330 immunostainer prior to operation.

a — diagram showing the correct placement of the slide in the instrument tray; b — slide protection device covering the slide with the sample and creating a humidity chamber effect.

 

To proceed with the next steps, it is necessary to select the optimal washing buffer. Phosphate-buffered saline (PBS) with a pH of 7.4 is typically used in histological studies. However, routine PBS is not recommended for IHC staining because it has high surface tension, which prevents the even distribution of reagents across the slide surface and reduces the efficiency of antibody penetration into the tissue. In such cases, buffers containing detergents, such as TBST (Tris-Buffered Saline with Tween-20) at pH 7.4, are the optimal solution. Alternatively, detergents may be added directly to the buffer solutions. For example, PBS with 0.5% Tween 20 exhibits properties similar to TBST and is a suitable washing buffer for IHC studies. An alternative to Tween 20 is another detergent — Triton X-100. However, it is important to note that Triton X-100 has more aggressive chemical properties and is more commonly used for cell lysis in immunocytochemical studies [7].

The deparaffinization step ensures more effective penetration of monoclonal antibodies into the tissue. Slides containing paraffin-embedded samples were washed three times in a deparaffinization buffer (Dartmоn) for 3 minutes at 72°C, followed by cooling to room temperature (20°C) . The samples were passed through a series of propanol solutions (100%, 95%, 85%, and 75%) (BioVitrum) for 40 minutes. To remove alcohols from the slides, they were washed three times with PBS-Tween 20 buffer (BioChemica), which is a necessary step for distributing the subsequently used reagents across the slide surface.

The epitope retrieval step facilitates the release of epitopes by breaking down the methylene bridges formed during aldehyde fixation. For 20 minutes, the slides were incubated in Tris-EDTA buffer with pH = 9 (DiaM) under conditions of elevated temperature up to 100°C. The temperature was then lowered to room temperature over 3 minutes. The instrument then washed the slides three times in PBS-Tween 20 buffer for 30 minutes at room temperature. After the epitope retrieval step, care must be taken to prevent the samples from drying out during all subsequent steps, as this may lead to the development of unwanted background staining.

Results

After removing the material from the Dartmon AS330 immunostainer (Dartmon), the samples were placed in a pre-prepared humid chamber to prevent them from drying out. The subsequent steps were performed manually (Fig. 2).

 

Fig. 2. The main reagents and equipment required for manual IHC staining.

To create a humidity chamber, we used two histology trays; on the bottom tray (which held the slides), we placed a sheet of filter paper moistened with water. The Dartmon detection system includes: a 3% H₂O₂ solution, anti-mouse/anti-rabbit goat antibodies, a conjugate of anti-goat antibodies with horseradish peroxidase, a chromogen (3,3’-diaminobenzidine), and a buffer for diluting the chromogen.

 

Peroxidase inhibition. Inhibition of endogenous peroxidase is necessary to prevent its interaction with the chromogen and the formation of a nonspecific signal.

In the first stage, endogenous peroxidase was blocked with 3% H₂O₂. An experiment was conducted to determine the optimal blocking time at three time points: 10, 20, and 30 minutes. It was found that the optimal incubation time for blocking endogenous peroxidase in lung tissue was 30 min (Fig. 3). After the incubation period, the slides were quickly rinsed in PBS-Tween 20 buffer (BioChemica).

 

Fig. 3. IHC staining of the ferret lung tissue with antibodies to the SARS-CoV-2 S protein following different incubation times of the samples with H₂O₂.

a–c — samples from animals in the control group; d–e — samples from animals infected with Omicron SARS-CoV-2. a, d — H₂O₂ incubation for 10 min; b, e — 20 min; c, f — 30 min.

Solid arrow — blood plasma in a pulmonary tissue vessel; dashed arrow — ciliated epithelium of a bronchiole. Nonspecific staining of both structures with the chromogen weakens as the incubation time of the preparations with H₂O₂ increases.

 

Protein block. Due to the lack of a universal immunostaining protocol, early studies were conducted without using bovine serum albumin (BSA) as a blocking agent. Most staining protocols are designed for medical applications. However, such protocols are often unsuitable for experimental studies aimed at visualizing viral markers. Consequently, most of them do not include data on the additional use of a blocking agent. As a result, when applying such staining methods in experiments to detect viral determinants, background staining associated with nonspecific antibody-antigen interactions was observed. Moreover, all medical studies use reagents approved by a state certificate, which is not a requirement for scientific research. Consequently, experimental studies require the selection of optimal conditions and reagents. It was experimentally determined that a protein blocker must be used in the study.

BSA, casein, or fish gelatin are commonly used as blocking agents. It is also permissible to use serum from the animal that is the host of the secondary antibody. Blocking agents are primarily proteins that bind to various epitopes on cells in tissue sections. Thus, this step helps reduce the percentage of nonspecific binding of the antibody to the antigen.

A previous study was conducted to determine the optimal BSA concentration: 0.5%, 1%, or 2% (Fig. 4). It was found that the optimal concentration for reducing background staining is at least 1%. The study also examined the blocking time and established that the required incubation time for samples with BSA is 40–50 min.

 

Fig. 4. Immunostaining of lung tissue from ferrets infected with Omicron SARS-CoV-2 using antibodies to the S protein, following incubation of samples with BSA at various concentrations.

a — in the absence of BSA; b — 0.5% BSA; c — 1% BSA; d — 2% BSA. Solid arrows — blood plasma in vessels; dashed arrows — ciliated epithelium layer. The proportion of nonspecific background staining in both structures decreases as the BSA concentration increases.

 

Antibody staining. The primary antibodies were applied immediately after the BSA treatment, without additional washing in buffer. The study used mouse monoclonal antibodies against the SARS-CoV-2 S protein (Hytest) at a concentration of 1 mg/mL. Monoclonal antibodies exhibit the highest specificity toward the viral determinants.

During the experimental work, it was found that BSA should be added to the antibody solution to achieve more representative staining. In addition to reducing background staining, BSA also acts as a preservative, contributing to the stabilization of antibodies and their longer storage in the buffer solution. The study examined two factors directly affecting staining quality: antibody concentration and BSA concentration. When diluting the antibodies 600-fold and 800-fold, 1% and 2% BSA concentrations were tested (Fig. 5). For this specific experiment, it was found that the optimal dilution was an 800-fold dilution of antibodies with the addition of 2% BSA. Minor background staining was observed under this condition in the form of nonspecific plasma staining; therefore, to obtain more representative staining, a 1000-fold dilution of antibodies with the addition of 2% BSA was used in subsequent steps of the experiment.

 

Fig. 5. IHC staining of lung tissue from animals infected with the Omicron strain of SARS-CoV-2, following incubation of samples with antibodies at various dilutions in combination with BSA at various concentrations.

a — ferret lung tissue stained with antibodies diluted 600-fold with the addition of 1% BSA; b — lung tissue stained with antibodies diluted 600-fold with the addition of 2% BSA; c — ferret lung stained with antibodies diluted 800-fold, with the addition of 1% BSA; d — ferret lung tissue stained with antibodies 800-fold, with the addition of 2% BSA.

 

Conjugation with secondary antibodies. The subsequent steps of IHC staining were performed using reagents included in the Dartmon detection system (cat. no. DMRD4044). After washing the slides in PBS buffer with Tween 20, a solution of goat antibodies specific to mouse and rabbit antibodies (Dartmon) was applied to the samples and incubated for 20 min, followed by washing in buffer. Next, a solution of anti-goat antibody conjugate with HRP (Dartmon) was applied to the slides and incubated for 20 minutes at room temperature. The instrument then washed the slides in PBS with Tween 20 for 10 minutes to remove antibodies that had not bound to the tissue.

Chromogen staining. To develop the signal marking the viral S-protein, the samples were incubated for 2 min with a solution of 3,3’-diaminobenzidine (DAB; Dartmon) — a horseradish peroxidase substrate (to prepare the working solution, 1 drop of DAB was dissolved in 1 mL of chromogen dilution buffer). After the incubation period, the slides were washed in PBS buffer with Tween 20.

Staining samples with hematoxylin. After completing all stages of immunostaining, the sections were stained with hematoxylin in the Tissue-Tek Prisma (Sakura Finetek) histology processor to visualize tissue structures. The parameters for the Mayer staining procedure [8] were entered into the Tissue-Tek Prisma (Sakura Finetek) system. The samples were incubated for 3 minutes in hematoxylin, followed by rinsing in distilled water for 1 minute. The sections were rinsed again in two portions of isopropanol, each for 1 minute. A final rinse was performed in two portions of xylene — 3 and 5 minutes, respectively. The sections were then placed in Tissue-Tek Film (Sakura Finetek) for automatic mounting of the stained sections under a cover slip.

Light microscopy. The obtained results were visualized on an Olympus VS 200 (Olympus) research slide scanner using a ×20 objective lens.

Imaging of specific staining of the SARS-CoV-2 S protein in lung tissue. Optimizing the IHC staining protocol makes it possible to reduce background signal and unambiguously determine the localization of specific viral markers. However, it is important to note that the intensity of specific staining depends on the affinity of the antibody to particular virus strains. Most modern monoclonal antibodies against the SARS-CoV-2 S protein have a relatively higher affinity for early strains (Alpha, Beta, Delta, Gamma) than for the later Omicron strain (Fig. 6). Nevertheless, this is not the only factor influencing the intensity of specific staining. Other important aspects include the infectious dose and the route of administration to experimental animals (intranasal, intratracheal, perfusion); the time elapsed since infection; and the animals’ immune status with respect to the viral markers under investigation.

 

Fig. 6. Imaging of specific IHC staining of lung tissue from ferrets infected with two different strains of SARS-CoV-2.

The localization of the SARS-CoV-2 S protein is marked by dark chromogen granules in the cytoplasm of the cells. Primary antibodies diluted 1,000-fold.

a — lung tissue from an animal infected with Delta SARS-CoV-2; b — lung tissue from an animal infected with Omicron SARS-CoV-2.

 

Discussion

The IHC staining method is widely used in virology [1]. IHC not only allows for the detection of viral antigens but also determines their localization within the tissue of interest and, in the case of multiplex analysis, the type of infected cells, which is valuable for scientific and medical research. However, the use of standard IHC protocols often leads to the denaturation of viral proteins in tissues [1, 9]. For this reason, there is a necessity to optimize the methodology for specific tasks.

In the present study, the SARS-CoV-2 S protein was examined as the antigen of interest. The first step in tissue preparation is fixation. When working with infectious agents, this step is also necessary to inactivate endogenous viral particles and ensure personnel safety. It has been shown that formalin leads to conformational changes in the S-protein trimer and the loss of infectivity of coronavirus particles [10]. Despite this, this method of fixation does not have a significant negative impact on the ability of the antigen to be detected during subsequent stages of sample preparation [11]. Thus, tissue fixation in formalin is the optimal approach both in terms of preserving endogenous determinants in the tissue and in terms of biosafety.

The accessibility of antigen for antibody is ensured through the following steps: deparaffinization of sections and retrieval of epitopes. Deparaffinization is necessary to extract the tissue from the embedding medium and increase its permeability to the washing/retrieval buffer. Tissue permeability is further enhanced by adding detergents to the washing buffers. Thus, Tween 20 is most commonly used in immunohistochemistry, while Triton X-100 remains less preferred due to its chemical aggressiveness [7]. Epitope retrieval can be performed in two ways: high-temperature or proteolytic. In the present study, SARS-CoV-2 S-protein retrieval was performed using the high-temperature method — samples were incubated in a high-pH buffer at 100°C. According to the literature, this approach is the most effective for extracting the coronavirus S-protein from tissue [12].

When working with horseradish peroxidase-conjugated antibodies, it is necessary to pre-block the tissue with 3% H₂O₂ to prevent the chromogen from interacting with endogenous peroxidase. In the present study, the optimal incubation time for sections with H₂O₂ was 30 minutes, although earlier publications reported shorter durations [11, 12]. Before applying primary antibodies, it is important to incubate the samples with a blocking agent; omitting this step may lead to nonspecific background staining. BSA, casein, or fish gelatin are commonly used as blocking agents. The use of serum from the animal host of the secondary antibody is also permitted [13]. In the proposed method, BSA was used as the blocking agent. The intensity of background staining depended on the concentration of BSA; the most optimal result was achieved when samples were incubated with 2% BSA. This concentration of the blocking agent was also used in the protocols of earlier studies [11].

The concentration of primary antibodies also has a significant impact on the quality of IHC staining. In the proposed protocol, monoclonal antibodies against the SARS-CoV-2 S-protein (Hytest, Cat. No. 3CV2) were used. The optimal dilution was determined experimentally and was 1:1000. To prevent background staining, 2% BSA was added to the aliquot. Subsequent stages of IHC staining were performed using the Dartmon detection system, which includes ready-to-use reagents. Although Dartmon products are widely used in medical research, data on the application of the aforementioned detection system for visualizing viral markers remain limited [14]. In the present study, we demonstrated the feasibility of using these reagents to visualize the S protein of the Omicron and Delta SARS-CoV-2 strains in ferret lung tissue.

When imaging samples from animals infected with the Delta and Omicron strains of SARS-CoV-2, a dark chromogen deposit localized in the cytoplasm of the cells was detected, corresponding to the S-protein. It is noteworthy that the structure of this protein is relatively variable (compared, for example, to the nucleoprotein) and differs depending on the coronavirus strain [15]. Consequently, the affinity of a single antibody variant for different variants of the S-protein can vary significantly, which may also affect the quality of IHC staining.

Conclusion

This study proposed an alternative method for staining ferret lung tissue for the SARS-CoV-2 S-protein (Delta and Omicron strains) using chromogenic immunohistochemistry.

Formalin fixation of tissue when working with SARS-CoV-2 infected material did not lead to denaturation of the viral S-protein epitopes and did not affect their ability to be visualized during subsequent stages of sample preparation.

Incubation of samples in Tris-EDTA buffer (pH 9) at 100°C for 20 min was proposed as an retrieval protocol ensuring effective recovery of coronavirus S-protein epitopes.

The effect of incubation time of samples with 3% H₂O₂ on the quality of IHC staining was demonstrated, and it was established that the optimal time for blocking endogenous peroxidase and achieving the lowest level of background staining was 30 min.

The intensity of background staining depended on the concentration of the blocking agent; BSA was used in this study, with an optimal concentration of 2%.

The results of staining using various concentrations of primary antibodies and BSA are presented, and an optimal dilution of 1:1000 with the addition of 2% BSA is recommended for monoclonal antibodies against the SARS-CoV-2 protein.

The possibility of successfully visualizing the SARS-CoV-2 S protein in lung tissue using the Dartmon detection system is demonstrated, although data on the use of these reagents for studying viral markers are not yet complete.

The IHC staining method is an effective tool for detecting viral antigens, determining their localization in tissue, and identifying infected cells, making it significant for virological and medical research. In this regard, there will be a necessity to optimize and develop new protocols.

 

¹ Recommendations of the EEC Board dated November 14, 2023, No. 33 "On the Guidelines for Working with Laboratory (Experimental) Animals during Preclinical (Non-Clinical) Studies." URL: https://docs.eaeunion.org/documents/415/7752/

² AVMA Guidelines for the Euthanasia of Animals: 2020 Edition. URL: https://www.avma.org/sites/default/files/2020-02/Guidelines-on-Euthanasia-2020.pdf

³ МMU 1.3.3103-13 "Organization of work of laboratories using methods of electron and atomic force microscopy in the study of cultures of microorganisms of pathogenicity groups I–IV."

About the authors

Ksenia F. Emtsova

State Scientific Center for Virology and Biotechnology “Vector”

Author for correspondence.
Email: k.emtsova@g.nsu.ru
ORCID iD: 0009-0003-5165-5357

research intern, Microscopic research department

Russian Federation, Koltsovo

Ekaterina V. Spiridonova

State Scientific Center for Virology and Biotechnology “Vector”

Email: spiridonova_ev@vector.nsc.ru
ORCID iD: 0009-0006-8655-6713

research intern, Microscopic research department

Russian Federation, Koltsovo

Vladimir V. Omigov

State Scientific Center for Virology and Biotechnology “Vector”

Email: omigov_vv@vector.nsc.ru
ORCID iD: 0000-0002-2028-6099

Cand. Sci. (Med.), leading researcher, Microscopic research department

Russian Federation, Koltsovo

Andrey S. Gudymo

State Scientific Center for Virology and Biotechnology “Vector”

Email: gudymo_as@vector.nsc.ru
ORCID iD: 0000-0001-6952-6412

researcher, Department of zoonotic infections and influenz

Russian Federation, Koltsovo

References

Supplementary files