Application of antibodies against Borna disease virus phosphoprotein and nucleoprotein on paraffin sections

  • Authors:
    • Ying Huang
    • Xiao‑Mei Li
    • Shi‑Gang Chen
    • Jing Deng
    • Yang Lei
    • Wen‑Juan Li
    • Hong‑Zhi Zhang
    • Hong Zhang
    • Dan Li
    • Peng Xie
  • View Affiliations

  • Published online on: January 22, 2018     https://doi.org/10.3892/mmr.2018.8467
  • Pages: 5416-5422
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Abstract

In order to study the application of antibodies against recombinant proteins for detecting Borna disease virus (BDV) phosphoprotein (p24) and nucleoprotein (p40) (BDV‑p24/p40) on paraffin sections by immunohistochemistry. The purified fusion p24 and p40 proteins were used for the preparation of polyclonal and monoclonal anti‑p24 and anti‑40 antibodies, which were confirmed by ELISA and western blotting. Paraffin sections were made from BDV‑infected Sprague‑Dawley (SD) rats (n=20), PBS‑injected SD rats (n=20), normal SD rats (n=20) and normal C57 mice (n=20). Immunohistochemical staining was performed according to the EnVision™ two‑step protocol. Heat‑mediated antigen retrieval was performed using the retrieval buffer sodium citrate (1 mM; pH 6.0). All the antibodies against recombinant proteins exhibited good sensitivity and specificity. There were significant differences between the BDV‑infected group and the BDV‑uninfected group for poly‑ and monoclonal anti‑p24 and ‑p40 antibodies. These antibodies against recombinant proteins may be used effectively to detect BDV p24 and p40 in paraffin sections.

Introduction

Borna disease virus (BDV) is an enveloped, non-segmented negative single-stranded RNA virus, which is highly neurotropic and is able to infect a range of warm-blooded animals, from birds to primates (13). Epidemiological investigations have demonstrated that neuropsychiatric disorders, including schizophrenia and mood disorders, are associated with BDV infection (411). Notably, BDV RNA and antigens have been detected in brain tissues from patients with psychiatric diseases and brain tumors (1217), in addition to in human lymphocytes (18).

The genome of BDV, at a length of 8.9 kb, consists of six open reading frames which respectively encode six proteins (19). Among these proteins, phosphoprotein (BDV p24) and nucleoprotein (BDV p40) are frequently detected together in human serological studies (2023); therefore, they were selected as the primary target antigens in the present study.

There are four principal methods of detecting BDV infection: i) Isolating virus from blood samples or tissue homogenate (5,8,14); ii) detecting BDV-RNA with reverse transcription-polymerase chain reaction (RT-PCR) analysis (24); iii) serological testing, including indirect immunological fluorescence assays (25), western blotting (26,27) and ELISA (7); and iv) detecting the primary antigens (p24 or/and p40) via immunohistochemical staining in situ (28). The first three methods require fresh specimens. Although serum or plasma samples are the easiest diagnostic sources, in clinical practice, pathological tissue from patients undergoing brain surgery has may be fixed and embedded into paraffin blocks for long-term preservation in pathology laboratories, which also are favorable materials for use in screening for BDV infection in the population via immunohistochemistry. Previous studies have used antibodies donated from other collaborating laboratories (29,30). However, there are no commercially available anti-BDV protein antibodies.

Monoclonal and polyclonal anti-p24 and anti-p40 antibodies have been prepared previously by animal immunization technology and hybridoma technology (31). Therefore, the primary purpose of the present study was to examine the application of the constructed antibodies against recombinant proteins for detecting p24 and p40 in paraffin sections of BDV-infected rat/mouse brain tissue by immunohistochemical staining (including appropriate dilutions, antigen retrieval and evaluation of the results). The present study may provide a foundation for detecting BDV infection in brain tissue paraffin sections, particularly from brain tumors, by immunohistochemistry (12,14).

Materials and methods

BDV strain

Human BDV strain Hu-H1 was isolated from the blood cells of a severely depressed patient with bipolar disorder in Germany, and co-cultured with a permanent human oligodendrocyte (OL) cell line (5). BDV-Hu-H1 (kindly supplied by Professor Hanns Ludwig, Free University of Berlin, Berlin, Germany) virus solution was obtained by repeated freezing and thawing of BDV-infected OL cells and stored at −80°C.

Viral infection
Infection of animals

Sprague-Dawley (SD) rats born within 24 h were intracranially inoculated in their right cerebral hemispheres with either: i) 30 µl Hu-H1 virus solution [104 focus forming units/ml; n=20; 11 males, weight (305±12 g); 9 females, weight (217±15 g)], as previously described (32); or ii) PBS [30 µl; negative control; n=20; males, weight (313±14 g); 12 females, weight (222±12 g)]. In addition, 20 SD rats without injection [10 males, weight (310±12 g); 10 females, weight (224±10 g)], and 20 uninjected C57 mice [11 males, weight (23±1 g); 9 females, weight (20±1 g)] as the normal control. Rats and mice were group-housed under a 12-h light/dark cycle (on between 7:00 a.m. and 7:00 p.m.) with unrestricted access to food and water at a constant room temperature of 22–24°C and 50% humidity. All animals that we used in this experiment were provided by the Experimental Animal Center at Chongqing Medical University (Chongqing, China). All the procedures performed in the present study were approved by the Ethics Committee of Chongqing Medical University (Chongqing, China).

Preparation of samples

All rats and mice were sacrificed on postnatal day 56. Each right brain hemisphere was collected and stored at −80°C. The total protein was extracted from the right brain tissue from the −80°C refrigerator (n=20) using the Total Protein Extraction kit (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China). The protein concentrations were determined with an Enhanced Bicinchoninic Acid Protein Assay kit (Beyotime Biotechnology Co., Ltd., Zhejiang, China). Protein samples (15 µg per lane) were subjected to SDS-PAGE on a 12% gel (100 V for 2 h) following heat denaturing at 95°C for 5 min. Proteins were electrophoretically transferred to polyvinylidene fluoride (PVDF) membranes (100 V for 75 min) and were blocked in 5% non-fat dried milk for 1 h at room temperature. The membranes were incubated overnight at 4°C with mouse anti-BDV nucleoprotein (p24) monoclonal antibody (1:5,000; kindly provided by Professor Hanns Ludwig) (31,32) and used anti-GAPDH antibody (1:5,000; ab9485, Abcam, Cambridge, UK) served as the loading control. The membranes were washed with TBS-Tween 20 (TBST), and further incubated with goat anti-mouse immunoglobulin (Ig)G-HRP (1:10,000; bs-0296G-HRP, Beijing Bioss Co., Ltd., Beijing, China) at room temperature for 2 h. Subsequent to washing the membranes, the protein bands were visualized using enhanced chemiluminescence detection reagents (Nanjing KeyGen Biotech Co., Ltd.). The signal was detected using the Bio-Rad ChemiDoc XRS system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Preparation of antibodies
Preparation of antigens

Full-length cDNAs of BDV p24 and p40 were generated using the RT-PCR (33). The PCR conditions were: An initial denaturation at 94°C for 5 min; 30 cycles of 94°CC for denaturation (40 sec), annealing at 60°C (40 sec) and elongation at 72°C (30 sec); and extension at 72°C for 5 min. Total RNAs isolated from BDV-Hu-H1-infected OL cells were used as templates. The primers used for RT-PCR were: 5′-TAACATATGACCATGCCACCCAAGAGACGC-3′ (forward) and 5′-CGCGGATCCAAGTTTAGACCAGTCACAC-3′ (reverse) for p40; and 5′-CCGCATATGGCAACGCGACCATCGAGT-3′ (forward) and 5′-CCGCTCGAGTGGTATGATGTCCCATTCATCC-3′ (reverse) for p24. The cDNAs of p24 and p40 were ligated into the vectors (Novagen; Merck KGaA, Darmstadt Germany) pET-41a by NdeI and XhoI sites and pET-14b by NdeI and BamHI sites, respectively. These vectors were transformed into Escherichia coli BL21 (DE3; the Key Laboratory of Neurobiology, Chongqing, China). The authenticity of the expression constructs was verified by sequencing. The recombinant histidine-tagged p24 and p40 proteins were used following purification using HisTrap HP (GE Healthcare, Chicago, IL, USA) column chromatography. The column was equilibrated with equilibration buffer and the bacterial lysate was filtered to obtain the supernatant, which was purified via a purification column. Unbound hybrid proteins were removed to obtain recombinant proteins by using wash buffer containing 20 mmol/l Tris-HCl (pH 8.0), 50 mmol/l imidazole and 500 mmol/l NaCl.

Polyclonal antibody preparation

Two adult male New Zealand white rabbits with the weight of 3 kg obtained from the Experimental Animal Center of Chongqing University were used for the experiment. Under specific pathogen-free conditions at a constant room temperature of 22–24°C and 50% humidity with a 12-h light/dark cycle (lights on: 8:00 a.m., off: 8:00 p.m.) and provided ad libitum access to food and water. The purified histidine-tagged p24 protein (500 µg diluted with 1 ml of physiological saline), was emulsified with an equal volume of complete Freund's adjuvant (Sigma-Aldrich, Merck KGaA) and injected intradermally into a rabbit over 10 to 12 sites. Following pre-immunization blood drawing, the rabbit received booster injections twice, at monthly intervals, with the same amount of protein mixed with incomplete Freund's adjuvant. Subsequent to the third injection, the serums were collected and purified by antigen immunoaffinity chromatography. The antibody against recombinant p40 was similarly produced. The activated agarose gel and antigen were coupled overnight to prepare an antigen-immobilized column. The closed column was pre-washed with PBS to equilibrate the column. Following, the serums were added to a well-balanced column and incubated overnight at 4°C and washed with the eluate to obtain purified antibody. The eluent was measured at 280 nm.

Monoclonal antibody preparation

A total of 5 BALB/c mice [female, 6–8 weeks old, weight (21±1 g)] were obtained from the Experimental Animal Center of Chongqing Medical University. Under specific pathogen-free conditions at a constant room temperature of 22–24°C and 50% humidity with a 12 h light/dark cycle (lights on: 8:00 a.m., off: 8:00 p.m.) and provided ad libitum access to food and water. The mice were first immunized intraperitoneally with histidine-tagged p24 protein (50 µg diluted with 100 µl saline, emulsified in Freund's complete adjuvant). Immunization was repeated three times at intervals of 2 weeks. Hybridoma was prepared by fusing SP2/0 myeloma cells (Nanjing GenScript Co., Ltd., Nanjing, China) and spleen cells of immunized BALB/c mice. Immunized mouse spleen cells and SP2/0 myeloma cells were hybridized with 50% PEG 4000 (Sigma-Aldrich Merck KGaA) at a 5:1 ratio and cultured in RPMI-1640 culture medium (Hyclone; GE Healthcare Life Sciences, Logan UT, USA). The recombinant BDV nucleus protein was used as the antigen, and the positive cell clone strains were measured by indirect ELISA (Cosmo ELISA Plate, 1105–096; Cosmo Biosciences, Inc., San Diego, CA, USA). The optical density (OD) of each well was measured at 450 nm. The criteria were as follows: OD was greater than or equal to 2.1X the negative control. Candidates with positive ELISA clones were subjected to limited dilution and further clones were screened. The hybridoma cell strains with stable secretory monoclonal antibody were determined by the limited dilution method for three-times sub clones with the preliminary screening of the suspected positive cell clones: The selected positive cell clones (1 cell/well) were seeded into a 96-well plate in HT medium (Hyclone; GE Healthcare Life Sciences) and were cultured for 4 weeks. The supernatant was then collected and detected via indirect ELISA, which was used until the positive rate was 100%. For the production of large amounts of monoclonal antibodies, mouse ascites of monoclonal antibodies were collected and purified by protein G affinity chromatography. Firstly, a purification column containing Sepharose-protein G was washed with binding buffer. Secondly, mouse ascites were diluted 4 times with binding buffer, and centrifuged at 11,900 × g for 30 min at 4°C. Thirdly, the supernatant was added into the washed purification column. The bound monoclonal antibodies were eluted with elution buffer and loaded in a collection tube containing 0.1 ml potassium phosphate buffer. Finally, the eluent was measured at A280 nm. The antibody against the recombinant p40 was similarly produced.

Titer and specificity of antibodies
Titer detection by ELISA

The recombinant proteins were diluted to a concentration of 100 ng/100 µl with 0.05 mol/l Na2CO3-NaHCO3 coating buffer (pH 9.6) and were added to 96-well plates (100 µl proteins/well). Following overnight incubation at 4°C, plates were washed with PBS-Tween 20 (PBST) three times. Wells were blocked with 5% bovine serum albumin (Hyclone; GE Healthcare Life Sciences) solution for 1 h at 37°C and washed with PBST three times. The pre-immunization serum was diluted 1:1,000 and the purified antibodies were diluted 1:1,000, and serially diluted two-fold from 1:1,000 to 1:512,000. The plates were incubated with 100 µl diluted antibodies for 1 h at 37°C. Wells were washed and further incubated with goat anti-rabbit immunoglobulin G antibody (Beijing Bioss Co., Ltd.) diluted 1:2,500 (100 µl/well), conjugated with horseradish peroxidase (HRP). Following washing of the plates, 3,3,5,5,-tetramethylbenzidine was added to each well. The chromogenic reaction was stopped by 50 µl/well 2 mol/l H2SO4 following a 10-min incubation at 37°C. The optical density of each well was measured at 450 nm. All samples were run in triplicate. PBS was used as a blank control and the pre-immunization serum was used as a negative control.

Specificity identification by western blotting

Proteins were extracted from normal OL cells and BDV-infected OL cells using the KeyGen Whole Cell Lysis Assay (Nanjing KeyGen Biotech Co., Ltd.). Protein concentrations were quantified according to the Bradford procedure (Bradford protein quantitation assay; Nanjing KeyGen Biotech Co., Ltd.) to maintain the same loads. Proteins (normal OL cells lysates, 25 µg; BDV-infected OL cell lysates, 25 µg; recombinant p24 and p40 BDV proteins, 1 µg) were separated by 12% SDS-PAGE (100 V for 2 h) following heat denaturation at 95°C for 5 min, and transferred onto PVDF membranes in Tris-glycine buffer containing 20% methanol (100 V for 75 min). The membranes were stripped according to marker bands and were blocked in 5% non-fat milk at room temperature for 1 h. The primary antibodies (1:1,500) were diluted with blocking buffer and incubated with membranes overnight at 4°C. The membranes were washed with TBST, and further incubated with HRP-conjugated secondary antibodies (1:2,000; bs-0295G-HRP and bs-0296G-HRP, Beijing Bioss Co., Ltd.) at room temperature for 2 h. Following washing of the membranes, the protein bands were visualized using enhanced chemiluminescence detection reagents (Nanjing KeyGen Biotech Co., Ltd.). The signal was detected using the Bio-Rad ChemiDoc XRS system (Bio-Rad Laboratories, Inc.). Additionally, the isolated recombinant BDV proteins by 12% SDS-PAGE were stained with Coomassie brilliant blue at room temperature for 3 h and then scanned by Epson Graphic Arts Scanner. The OD of each band was semi-quantified using Quantity One software (version 4.4.0; Bio-Rad Laboratories, Inc.).

Immunohistochemical analysis
Paraffin section preparation

All rats and mice were sacrificed on postnatal day 56. All left-brain hemispheres were fixed with 4% paraformaldehyde at room temperature for 24 h and embedded in paraffin. Tissue section was sliced along the sagittal plane into 5-µm sections, for the purpose of immunohistochemical staining.

All tissue sections from BDV-infected rats (n=20), PBS-injected rats (n=20), normal SD rats (n=20) and normal C57 mice (n=20) were tested with four antibodies against recombinant proteins respectively: Poly- and monoclonal anti-p24 antibodies (pAbp24 and mAbp24), and poly- and monoclonal anti-p40 antibodies (pAbp40 and mAbp40). All primary antibodies (pAbp24, mAbp24, pAbp40 and mAbp40) were diluted 1:50, 1:100, 1:200, 1:400, 1:600, 1:800, 1:1,000, 1:1,200, 1:1,400, 1:1,600, 1:1,800, 1:2,000 and 1:5,000.

Immunohistochemical staining

The technique of detection of BDV in the brain by immunohistology was pioneered by Gosztonyi et al (34). Immunohistochemical staining was performed according to the EnVision two-step protocol (EnVision; Dako; Agilent Technologies, Inc., Santa Clara, CA, USA). Varying concentrations of alcohol used for rehydration were as follows: Two washes with absolute ethanol 3 min, 95% ethanol 3 min, 85% ethanol 3 min and 80% ethanol 3 min. Heat-mediated antigen retrieval was performed within 3 min using retrieval buffer sodium citrate (1 mM; pH 6.0) with a pressure cooker. Then the slices were washed with PBS for 3 min, three times and blocked with 3% hydrogen peroxide at room temperature for 10 min. The primary antibodies were incubated overnight at 4°C and the blank control was treated with PBS instead of primary antibody, overnight at 4°C. Following incubation with HRP-conjugated secondary antibody (K406511-2, EnVision™; Dako; Agilent Technologies, Inc.) at room temperature for 30 min, the sections were stained with diaminobenzidine for a few sec and counterstained with hematoxylin for 3 min at room temperature.

Evaluation criteria

The present study focused on the evaluation of the intensity of positive staining, positive proportion of neurons and neuroglial cells and background staining in three brain regions: Cerebral cortex, hippocampus and cerebellum. p24 is has been demonstrated to exhibit strong positive staining in the cytoplasm and weak staining in the nucleus; p40 has been demonstrated to exhibit strong positive staining in the nucleus and weak staining in the cytoplasm (35). Positive staining was graded according to the intensity and proportion. For intensity: 0, no positive cells; 1, light yellow; 2, brown-yellow; and 3, tan. For proportion: 0, no positive cells; 1, 1–25%; 2, 26–50%; 3, 51–75%; and 4, 76–100% (36).

Statistical analysis

A total of 10 representative areas were selected under a high magnification field (×400) for each slide. The mean intensity score was multiplied by the mean proportion score to produce the total score under all the dilution levels of the four primary antibodies, for BDV-infected and uninfected (negative control and normal control) groups, respectively. The total scores between the two groups were tested using the non-parametric Mann-Whitney test in SPSS (version 21.0; IBM Corp., Armonk, NY, USA). Data are presented as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.

Results

Viral infection

The infection of 20 BDV-infected rats were confirmed by western blotting with monoclonal anti-p24 antibody (kindly provided by Professor Hanns Ludwig) (Fig. 1).

Purification of recombinant proteins

The purity of the recombinant p24 and p40 were analyzed by SDS-PAGE on a 12% gel (Fig. 2). The recombinant protein was primarily expressed in soluble form and purified by affinity chromatography. The band of His-p24 was ~24 kDa with a purity of 85%, and the band of His-p40 appeared at ~40 kDa with a purity of 90%. Results were confirmed by western blotting with monoclonal antibodies against p24 and p40, respectively (donated by Professor Hanns Ludwig), and the reaction bands were observed at 24 and 40 kDa. The results of western blotting demonstrated that the purified recombinant p24 or p40 protein was specifically bound to the monoclonal antibody. It was, therefore, immunologically confirmed that the expressed recombinant proteins had the correct antigenic activity and were able to be used to detect the antibody constructed.

Titer of antibodies

Positive dilutions were determined when the absorbance ratio of antibodies to the negative control was >2:1 (Fig. 3). The data demonstrated that the titer of the polyclonal anti-p24 antibody was 1:128,000, as well as the monoclonal antibody. The titer of polyclonal and monoclonal anti-p40 antibody was 1:256,000 and 1:128,000, respectively.

Western blot analysis

Western blot analysis demonstrated that proteins from BDV-infected OL cells and recombinant proteins were detected positively by pAbp24, mAbp24, pAbp40 and mAbp40 (Fig. 4). All normal OL cells exhibited no signal. Pre-immunization sera were used as the control, instead of primary antibodies, and there was no signal for either the recombinant or native proteins (data not shown). The results showed that the four antibodies had a good specificity for BDV p24 and p40, respectively.

Immunohistochemical analysis

The results of the four primary antibodies (pAbp24, mAbp24, pAbp40 and mAbp40) were positive in the BDV-infected group (n=20) and were negative in the BDV-uninfected group (n=60) (Figs. 5 and 6). For polyclonal anti-p24 and anti-p40 antibodies, the dilutions of 1:100 to 1:400 obtained strong positive staining with slight background coloring, and were therefore considered to be a suitable dilution. For monoclonal anti-p24 and anti-p40 antibodies, the dilutions of 1:200 to 1:400 obtained strong positive staining without background coloring, and were therefore considered to be a suitable dilution. The total mean scores for the four antibodies were 8 (mean) and 0 (mean) in BDV-infected and uninfected group, respectively. P-values (P=0.000) were generated by the nonparametric Mann-Whitney test between the BDV-infected and uninfected group and the mean ± standard deviation of the BDV-infected group was 7.38±3.59, and 0.07±0.28 for the uninfected group.

Discussion

There are no commercially available antibodies for BDV p24 and p40 on the market. In the present study, recombinant full-length BDV p24 and p40 were used as antigens to immunize rabbits and mice. Antibodies usually only recognize specific epitopes with conformational structures. Since in western blotting the antigen is denatured into a linear structure, this may lead to different results compared with methods using the conformational antigen and antibody (37,38). The issue of conformational epitopes on BDV-p24/p40 has been addressed. The conformational epitopes of the donated monoclonal antibodies used in the present study and their characterization have been examined previously (31,39). However, the conformational epitopes of the antibodies against recombinant proteins produced in the present study remain to be elucidated, and further analysis is required. However, the high specificity and sensitivity of the four antibodies produced in the present study were confirmed by western blotting, ELISA analysis and immunohistochemistry on paraffin sections.

Analysis of BDV protein expression (particularly p24/p40) in human brain tissue may determine BDV infection in humans; however, this method is unsuitable for epidemiological screening due to the low viral titer in patients and the necessity for time-consuming surgery. Serum or plasma samples are the easiest diagnostic sources and infection may be determined by easily accessible blood samples. As BDV is a highly neurotropic virus, brain tissue represents an ideal sample source to detect BDV infection. Researchers have observed BDV RNA and antigens in the brains of patients with neuropsychiatric disorders (14,40). Clinical human brain tissue samples are frequently stored in paraffin blocks; therefore, it may be convenient to detect BDV infection in these samples by immunohistochemistry, and to study the correlation between BDV infection and morbidity (40).

Compared with the polyclonal antibodies in the present study, the sensitivity of the monoclonal antibodies was consistent and the specificity was increased; additionally, the nonspecific staining of the background was decreased with the monoclonal antibodies. The results of the present study demonstrated that the polyclonal and monoclonal anti-p24 and anti-p40 antibodies against recombinant proteins were highly specific and sensitive for BDV p24 and p40 detection, and may be efficiently used to detect BDV infection in paraffin sections of human brain tissue in future studies.

Acknowledgements

The authors of the present study would like to thank Professor Liv Bode and Professor Hanns Ludwig of the Robert Koch Institute and Institute of Virology at the Free University of Berlin (Berlin, Germany) for providing the human oligodendroglioma cell line and the Borna disease virus strain Hu-H1 used in the present study. The present study was supported by the National Basic Research Program of China (973 Program) (grant no. 2009CB918300) and the Natural Science Foundation of Chongqing City of China (grant no. cstc2013jcyjA10003).

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April 2018
Volume 17 Issue 4

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Online ISSN:1791-3004

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APA
Huang, Y., Li, X., Chen, S., Deng, J., Lei, Y., Li, W. ... Xie, P. (2018). Application of antibodies against Borna disease virus phosphoprotein and nucleoprotein on paraffin sections. Molecular Medicine Reports, 17, 5416-5422. https://doi.org/10.3892/mmr.2018.8467
MLA
Huang, Y., Li, X., Chen, S., Deng, J., Lei, Y., Li, W., Zhang, H., Zhang, H., Li, D., Xie, P."Application of antibodies against Borna disease virus phosphoprotein and nucleoprotein on paraffin sections". Molecular Medicine Reports 17.4 (2018): 5416-5422.
Chicago
Huang, Y., Li, X., Chen, S., Deng, J., Lei, Y., Li, W., Zhang, H., Zhang, H., Li, D., Xie, P."Application of antibodies against Borna disease virus phosphoprotein and nucleoprotein on paraffin sections". Molecular Medicine Reports 17, no. 4 (2018): 5416-5422. https://doi.org/10.3892/mmr.2018.8467