key: cord-016858-pbjj50bx authors: Jung, Kwonil title: Immunohistochemical Staining for Detection of Porcine Epidemic Diarrhea Virus in Tissues date: 2015-09-10 journal: Animal Coronaviruses DOI: 10.1007/978-1-4939-3414-0_2 sha: doc_id: 16858 cord_uid: pbjj50bx Porcine epidemic diarrhea virus (PEDV), a member of the genus Alphacoronavirus, has resulted in significant economic losses in the European, Asian, and North American swine industries in previous years. PEDV infection causes acute diarrhea/vomiting, dehydration, and high morbidity and mortality in seronegative neonatal piglets. In this chapter, materials and methods for performing immunohistochemistry (IHC) for the detection of PEDV antigens in frozen or formalin-fixed, paraffin-embedded (FFPE) tissues are provided. In IHC of frozen tissues where viral antigens are well preserved, the use of specific antibodies labeled with fluorescence dyes provides excellent advantages and convenience, resulting in high sensitivity and specificity of IHC and reduction of operation time. In IHC of FFPE tissues where tissue or cell morphology is well preserved, the use of specific antibodies labeled with enzymes, such as alkaline phosphatase, also gives rise to significant advantages in defining the correlation of viral antigens with histopathologic lesions. PEDV antigens in frozen tissues are visualized as green staining in the cytoplasm of infected cells by fluorescent dyes conjugated with antibodies when activated by exciting light of a specific wavelength under a fluorescence microscope. In FFPE tissues, PEDV antigens are visualized as red staining in the cytoplasm of infected cells by the deposition of the substrate chromogen, Fast Red. 2013 to present, has led to a substantial loss of piglets (more than 10 % of US swine population). Because of similar clinical and pathogenic features between PEDV and another Alphacoronavirus , transmissible gastroenteritis virus (TGEV), or a Deltacoronavirus , porcine deltacoronavirus (PDCoV), differential laboratory tests are required for their diagnosis [ 3 -5 ] . Reverse transcriptionpolymerase chain reaction ( RT-PCR ) or quantitative RT-PCR (qRT-PCR) is useful for the rapid differential diagnosis; however, detection of viral antigens in tissues is essential for confi rming each viral infection . Viral antigens in frozen, fi xed cells, or tissues can be detected by immunohistochemistry (IHC) (or immunohistochemical staining) using specifi c antibodies labeled with fl uorescent dyes, such as Alexa Fluor ® 488 and fl uorescein isothiocyanate (FITC), or enzymes, such as alkaline phosphatase (AP) and peroxidase. Immunofl uorescence (IF) staining is the fi rst immunohistochemical staining method but is still widely used in veterinary and medical diagnosis . With fundamentality of antigen -antibody and antibody-antibody binding reactions, antigens are visualized by fl uorescent dyes conjugated with antibodies when activated by an exciting light of a specifi c wavelength (499-519 nm for Alexa Fluor ® 488 and 494-521 nm for FITC), under a fl uorescence microscope. Due to the high sensitivity, specifi city, and convenience in using Alexa Fluor ® 488 in frozen tissues, this chapter details an IF staining method for the rapid and precise detection of PEDV antigens in PEDV-infected, frozen tissues, contributing to verifi cation of the tissue sites of PEDV replication in infected pigs. A combination of PEDV-specifi c anti-sera as antigen detection antibody and secondary antibody conjugated with Alexa Fluor ® 488 is applied in the IF staining method. However, because of suboptimal conditions of tissue or cell morphology in frozen tissues, this staining method limits investigation of the correlation of PEDV antigens with histopathologic lesions. To compensate for the limitation of IF staining in frozen tissues, additional IHC staining method using enzyme-labeled antibodies, i.e., immunoenzymological staining, in formalin-fi xed, paraffi n-embedded (FFPE) tissues is also provided in this chapter. After adding a substrate of enzyme, such as Fast Red, it generates insoluble particles that can be localized in cells or tissues under light microscope. Compared to the IF staining in frozen tissues, the IHC in FFPE tissues has more accurate localization of antigens with a better contrast ratio, contributing to defi ning the correlation of PEDV antigen -positive cells with severity of histopathologic lesions caused by PEDV, such as intestinal villous atrophy. Only one serotype of PEDV has been reported from different countries [ 4 ] . There has been no evidence of cross-reactivity of PEDV with TGEV [ 6 , 7 ] . The use of hyperimmune anti-sera or polyclonal antibodies against PEDV in IHC staining is likely able to detect geographically different strains of PEDV and differentiate them from TGEV in tissues [ 8 , 9 ] , but with a potential disadvantage of inducing background or false signals. To improve the sensitivity and specifi city of IHC for the detection of PEDV antigens in tissues, the use of monoclonal antibodies to structural proteins of PEDV, such as spike (S) or membrane (M) protein, has been preferred [ 10 , 11 ] . Prior to their application on the tissues, potential cross-reactivity of monoclonal antibodies to TGEV and PDCoV can be tested by more sensitive assays, such as enzyme-linked immunosorbent assay, immunoblotting, and immunoprecipitation, compared to IHC. Tissue tropism of PEDV is related to the expression of aminopeptidase N (APN), a 150 kDa glycosylated transmembrane protein identifi ed as the cellular receptor , on porcine small intestinal villous enterocytes [ 12 ] . PEDV-infected enterocytes rapidly undergo acute necrosis, leading to marked villous atrophy in the small but not large intestine [ 10 ] . In PEDV-infected nursing pigs, major histologic lesions include acute diffuse, severe atrophic enteritis , and mild vacuolation of superfi cial epithelial cells and subepithelial edema in cecum and colon [ 8 -10 , 13 ] . PEDV antigens are observed mainly in villous enterocytes of the small (duodenum to ileum) and large intestines (except rectum) [ 1 , 8 , 10 ] . Occasionally, a few PEDV-positive cells were detected in the intestinal crypt cells or the Peyer's patches [ 1 , 8 -10 ] . No lesions were seen in the spleen, liver, lung, kidney, and mesenteric lymph node of orally and/or intranasally infected piglets [ 8 ] . Lung tissue of oronasally infected pigs was negative for PEDV antigen [ 1 , 8 -10 ] . PEDV antigens were not detected in other major organs, such as the pylorus, tonsils, liver, and kidneys. However, a recent study reported the replication of PEDV in swine pulmonary macrophages in vitro and in vivo [ 14 ] . Epidemic PEDV strains are highly enteropathogenic and acutely infect villous epithelial cells of the entire small and large intestines, but the jejunum and ileum are the primary sites of infection . To detect PEDV antigen in tissues and evaluate the pathogenicity of PEDV strains in pigs, the jejunum and ileum are the most critical tissue samples for performing IHC. 2. Place the slides in the rack into xylene for 20 min at RT to remove the paraffi n. 3. Place the slides in 100 % ethanol for 5 min to rehydrate tissues through a graded ethanol series (100-50 %; the following steps Only) (200-300 μl) and incubate in a humidifi ed chamber at 37 °C for 1 h ( see Note 3 ). 10 . Rinse the slides gently with PBS on a rocker platform shaker at RT for 5 min. Repeat through three changes of fresh PBS, 5 min for each. 11 . Gently blot the slides with Kimwipes around the tissue and place horizontally. 12. Apply 3-5 drops of Prolong ® Gold Antifade Mountant (with DAPI) to the tissue section using a glass dropping pipette and immediately put a cover slip on ( see Note 4 ). 13. The slides are ready to be evaluated under fl uorescence microscope. They need to be stored in a dark area until evaluation. PEDV antigens will appear to be green or as fl uorescent staining in the cytoplasm of infected cells (Fig. 1 ) . Cell nuclei are stained blue with DAPI. 1. When antigen retrieval procedure is completed (Sect. 3.3 ), drop 300-500 μl of 1× Universal Blocking Reagent on the tissue within the hydrophobic barrier and incubate at 37 °C for 30 min. 2. Drain the slides and place them on a horizontal surface. 8. Drain the slides and place them on a horizontal surface. 9. Apply the Fast Red solution enough to cover the tissue section (300-500 μl) and incubate in a humidifi ed chamber at RT for 30-60 min ( see Note 6 ). 10. Place the slides in a rack and rinse well in distilled water. Three changes, 2 min each. 11. Tissue sections are counterstained in a glass dish with Gill's hematoxylin at RT for 10 min. 12. Rinse the slides thoroughly in tap water for 5 min, and move into deionized water. 13. Drain the slides and place them on a horizontal surface. 14. Apply 2-4 drops of Permanent Aqueous Mounting Medium to the tissue section ( see Note 7 ), and immediately put a cover slip on ( see Note 8 ). 15. The slides are ready to be evaluated under light microscope. PEDV antigens will appear as a red precipitate in the cytoplasm of infected cells (Fig. 2 ) . Cell nuclei are stained blue with hematoxylin. 1. Use of fresh reagents is recommended. A large amount of washing buffer, 1× PBS, is needed, because complete washing is critical to reduce background and increase true signals. 2. Throughout immunostaining procedures, the tissues should stay rehydrated. Adequate antigen -antibody or antibody-antibody binding reaction is not expected in dried tissues, resulting in poor or weak staining results or a high level of background staining. It is also critical for a comparative immunostaining study in multiple different tissues. 3. The optimal dilutions of primary and secondary antibodies should be tested and selected in both frozen and FFPE tissue conditions. 4. Instead of plastic pipette tips, the use of glass dropping pipette will reduce the number of bubbles in the mounting medium as applied to the tissue sections. 5. When the Fast Red tablet is completely dissolved, the solution can be fi ltered via 0.9 μm syringe fi lter and used to reduce an irregular deposition of Fast Red on the tissues or background. 6. The color development, including intensity of true or false signals, in all tissue slides tested should be frequently monitored under the microscope. Wipe the non-charged slide surface with Kimwipes before putting the tissue slides on the microscope. 7. Gently drop the mounting medium so as not to create bubbles. The mounted slides need to be evaluated as soon as possible, because bubbles can be created spontaneously in the mounted medium. Detection of PEDV antigens ( red staining) in the cytoplasm of enterocytes lining atrophied villi by immunohistochemical staining in formalin-fi xed, paraffi nembedded jejunal tissues using a monoclonal antibody specifi c for the spike protein of PEDV and secondary antibody conjugated with alkaline phosphatase. Original magnifi cation ×200. Immunohistochemistry. Fast Red. Gill's hematoxylin counterstaining 8. To make the stained slides permanent, a large amount of mounting medium can be applied to the tissues so that the entire section is covered. Place slides horizontally in a 60 °C oven for 30 min to allow the medium to harden. Remove the slides from the oven, and allow them to cool at RT. Dip the slides in xylene and cover slip with permount permanent mounting medium (Fisher Scientifi c). 10% (w/v) Sucrose solution Antifade Mountant with 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI) (Invitrogen) 1× Phosphate-buffered saline (PBS) (pH 7.4) 1× PBS (pH 7.4) containing Tween 20, 0.1 % 100, 95, 70, and 50 % ethanol % (v/v) glacial acetic acid in deionized water 1× PBS 1× PBS (pH 7.4) containing Tween 20, 0.1 % M Tris buffer Gill's or Mayer's hematoxylin Ultramount Permanent Aqueous Mounting Medium (Dako) Glass slide-staining dishes or jars. 2. Slide racks and trays Adjustable pipettors with tips Barrier Dako Pen (Dako) Microscope slides: Superfrost™ Plus Gold Slides Glass cover slip (Fisher Scientifi c) Emergence of Porcine epidemic diarrhea virus in the United States: clinical signs, lesions, and viral genomic sequences Fighting a deadly pig disease Porcine epidemic diarrhea virus infection: Etiology, epidemiology, pathogenesis and immunoprophylaxis Diseases of swine Pathogenicity of 2 porcine deltacoronavirus strains in gnotobiotic pigs Quantitation, biological and physicochemical properties of cell culture-adapted porcine epidemic diarrhea coronavirus (PEDV) An immunoelectron microscopic and immunofl uorescent study on the antigenic relationship between the coronavirus-like agent, CV 777, and several coronaviruses The pathogenesis of an enteric infection in pigs, experimentally induced by the coronavirus-like agent, Cv-777 An immunohistochemical investigation of porcine epidemic diarrhoea Pathology of US porcine epidemic diarrhea virus strain PC21A in gnotobiotic pigs Monoclonal antibody-based immunohistochemical detection of porcine epidemic diarrhea virus antigen in formalin-fi xed, paraffi n-embedded intestinal tissues Porcine aminopeptidase N is a functional receptor for the PEDV coronavirus Pathology of experimental CV777 coronavirus enteritis in piglets. I. Histological and histochemical study Porcine epidemic diarrhea virus infects and replicates in porcine alveolar macrophages Salaries and research support were provided by state and federal funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University. This work was supported by a grant from the OARDC SEEDS, Grant # OAOH1536.