Sensitive and Specific Immunohistochemistry Protocol for Nucleocapsid Protein from All Common SARS-CoV-2 Virus Strains in Formalin-Fixed, Paraffin Embedded Tissues

Human coronavirus disease 2019 (COVID-19) is a life-threatening and highly contagious disease caused by coronavirus SARS-CoV-2. Sensitive and specific detection of SARS-CoV-2 viral proteins in tissues and cells of COVID-19 patients will support investigations of the biologic behavior and tissue and cell tropism of this virus. We identified commercially available affinity-purified polyclonal antibodies raised against nucleocapsid and spike proteins of SARS-CoV-2 that provide sensitive and specific detection of the virus by immunohistochemistry in formalin-fixed, paraffin-embedded tissue. Two immunohistochemistry protocols are presented that are mutually validated by the matched detection patterns of the two distinct viral antigens in virus-infected cells within autopsy lung tissue of COVID-19 deceased patients. Levels of nucleocapsid protein in the lungs of COVID-19 decedents, as measured by quantitative histo-cytometry of immunohistochemistry images, showed an excellent log–linear relationship with levels of viral nucleocapsid RNA levels, as measured by qRT-PCR. Importantly, since the nucleocapsid protein sequence is conserved across all known viral strains, the nucleocapsid immunohistochemistry protocol is expected to recognize all common variants of SARS-CoV-2. Negative controls include autopsy lung tissues from patients who died from non-COVID-19 respiratory disease and control rabbit immunoglobulin. Sensitive detection of SARS-CoV-2 in human tissues will provide insights into viral tissue and cell distribution and load in patients with active infection, as well as provide insight into the clearance rate of virus in later COVID-19 disease stages. The protocols are also expected to be readily transferable to detect SARS-CoV-2 proteins in tissues of experimental animal models or animals suspected to serve as viral reservoirs.

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Sensitive and specific immunohistochemistry protocols for detection of SARS-CoV-2 nucleocapsid and spike proteins in formalin-fixed, paraffin-embedded COVID-19 patient tissues

10.21203/rs.3.pex-1011/v1 ◽

2020 ◽

Author(s):

Yunguang Sun ◽

Linna Ge ◽

Mary J. Rau ◽

Mollie D. Patton ◽

Alexander J. Gallan ◽

...

Keyword(s):

Lung Tissue ◽

Polyclonal Antibodies ◽

Specific Detection ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Life Threatening ◽

Negative Controls ◽

And Control ◽

Formalin Fixed ◽

Spike Proteins

Abstract Human coronavirus disease 2019 (COVID-19) is a life-threatening and highly contagious disease caused by coronavirus SARS-CoV-2. Sensitive and specific detection of SARS-CoV-2 virus in tissues and cells of COVID-19 patients will support investigations of the biologic behavior and tissue and cell tropism of this virus. We identified two commercially available affinity-purified polyclonal antibodies raised against Nucleocapsid and Spike proteins of SARS-CoV-2 that provide sensitive and specific detection of the virus by immunohistochemistry in formalin-fixed, paraffin-embedded tissue. Protocols are presented that are mutually validated by matched detection patterns of virus-infected cells in autopsy lung tissue of COVID-19 deceased patients by the two distinctly different antibodies. Negative controls include autopsy lung tissue from patient who died from non-COVID-19 respiratory disease and control rabbit immunoglobulin. SARS-CoV-2 detection in human tissues will provide insights into viral tissue and cell distribution and load in patients with active infection as well as provide insight into clearance of virus in late COVID-19 disease stages.

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Monoclonal and Polyclonal Antibodies for Immunohistochemical Detection of Bovine Parainfluenza Type 3 Virus in Frozen and Formalin-Fixed Paraffin-Embedded Tissues

Journal of Veterinary Diagnostic Investigation ◽

10.1177/104063879200400404 ◽

1992 ◽

Vol 4 (4) ◽

pp. 393-399 ◽

Cited By ~ 8

Author(s):

Deborah M. Haines ◽

Jane C. Kendall ◽

Brad W. Remenda ◽

Michelle M. Breker-Klassen ◽

Edward G. Clark

Keyword(s):

Polyclonal Antibodies ◽

Accurate Identification ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Fresh Frozen ◽

Type 3 ◽

Murine Monoclonal Antibodies ◽

Formalin Fixed ◽

Immunohistochemical Testing

Accurate identification of bovine Parainfluenza type 3 virus in bovine respiratory disease requires dependable, sensitive, and specific techniques for detection in affected animals. Immunohistochemical testing can be a rapid and reliable means of demonstration of virus in tissues from suspect cases; however, this procedure is dependent upon the quality of the antisera directed against the viral antigens. The production of rabbit polyclonal and murine monoclonal antibodies directed against bovine Parainfluenza type 3 virus and techniques for their use in fresh-frozen and formalin-fixed paraffin-embedded tissues in immunofluorescence and immunoperoxidase-based immunohistochemical tests are described.

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Detection of Streptococcus Suis by in Situ Hybridization, Indirect Immunofluorescence, and Peroxidase-Antiperoxidase Assays in Formalin-Fixed, Paraffin-Embedded Tissue Sections from Pigs

Journal of Veterinary Diagnostic Investigation ◽

10.1177/104063870001200305 ◽

2000 ◽

Vol 12 (3) ◽

pp. 224-232 ◽

Cited By ~ 16

Author(s):

Mette Boye ◽

Anne A. Feenstra ◽

Conny Tegtmeier ◽

Lars Ole Andresen ◽

Søren R. Rasmussen ◽

...

Keyword(s):

In Situ Hybridization ◽

Indirect Immunofluorescence ◽

Polyclonal Antibodies ◽

Single Cells ◽

Streptococcus Suis ◽

Tissue Sections ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Formalin Fixed

Streptococcus suis is an important pathogen in pigs and is considered a zoonotic agent. To aid diagnosis of infection caused by S. suis, a species-specific probe targeting 16S ribosomal RNA was designed and used for fluorescent in situ hybridization. Two additional immunohistochemical detection methods, an indirect immunofluorescence assay and a peroxidase-antiperoxidase method, using polyclonal antibodies also were developed. The specificity of the oligonucleotide probe was examined by whole-cell and dot-blot hybridization against reference strains of the 35 serotypes of S. suis and other closely related streptococci and other bacteria commonly isolated from pigs. The probe was specific for S. suis serotypes 1–31. The specificity of the polyclonal antibodies, which has previously been evaluated for use in diagnostic bacteriology for typing of serotype 2, was further evaluated in experimentally infected murine tissue with pure culture of different serotypes of S. suis, related streptococci, and other bacteria commonly found in pigs. The polyclonal antibodies against S. suis serotype 2 cross-reacted with serotypes 1 and 1/2 in these assays. The in situ hybridization and the immunohistochemical methods were used for detection of S. suis in formalin-fixed, paraffin-embedded tissue sections of brain, endocardium, and lung from pigs infected with S. suis. The methods developed were able to detect single cells of S. suis in situ in the respective samples, whereas no signal was observed from control tissue sections that contained organisms other than S. suis. These techniques are suitable for determining the in vivo localization of S. suis for research and diagnostic purposes.

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Multi-omic analyses in Abyssinian cats with primary renal amyloid deposits

Scientific Reports ◽

10.1038/s41598-021-87168-0 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Francesca Genova ◽

◽

Simona Nonnis ◽

Elisa Maffioli ◽

Gabriella Tedeschi ◽

...

Keyword(s):

Renal Amyloidosis ◽

Amyloid Deposits ◽

Formalin Fixed Paraffin ◽

Familial Form ◽

Formalin Fixed Paraffin Embedded ◽

And Function ◽

Exclusive Or ◽

And Control ◽

Formalin Fixed ◽

Omics Data Integration

AbstractThe amyloidoses constitute a group of diseases occurring in humans and animals that are characterized by abnormal deposits of aggregated proteins in organs, affecting their structure and function. In the Abyssinian cat breed, a familial form of renal amyloidosis has been described. In this study, multi-omics analyses were applied and integrated to explore some aspects of the unknown pathogenetic processes in cats. Whole-genome sequences of two affected Abyssinians and 195 controls of other breeds (part of the 99 Lives initiative) were screened to prioritize potential disease-associated variants. Proteome and miRNAome from formalin-fixed paraffin-embedded kidney specimens of fully necropsied Abyssinian cats, three affected and three non-amyloidosis-affected were characterized. While the trigger of the disorder remains unclear, overall, (i) 35,960 genomic variants were detected; (ii) 215 and 56 proteins were identified as exclusive or overexpressed in the affected and control kidneys, respectively; (iii) 60 miRNAs were differentially expressed, 20 of which are newly described. With omics data integration, the general conclusions are: (i) the familial amyloid renal form in Abyssinians is not a simple monogenic trait; (ii) amyloid deposition is not triggered by mutated amyloidogenic proteins but is a mix of proteins codified by wild-type genes; (iii) the form is biochemically classifiable as AA amyloidosis.

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Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections.

Journal of Histochemistry & Cytochemistry ◽

10.1177/39.6.1709656 ◽

1991 ◽

Vol 39 (6) ◽

pp. 741-748 ◽

Cited By ~ 1765

Author(s):

S R Shi ◽

M E Key ◽

K L Kalra

Keyword(s):

Polyclonal Antibodies ◽

Antigen Retrieval ◽

Fixed Tissue ◽

Formalin Fixed Tissue ◽

Tissue Sections ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Pre Treatment ◽

Immunohistochemical Techniques ◽

Formalin Fixed

We describe a new approach for retrieval of antigens from formalin-fixed, paraffin-embedded tissues and their subsequent staining by immunohistochemical techniques. This method of antigen retrieval is based on microwave heating of tissue sections attached to microscope slides to temperatures up to 100 degrees C in the presence of metal solutions. Among 52 monoclonal and polyclonal antibodies tested by this method, 39 antibodies demonstrated a significant increase in immunostaining, nine antibodies showed no change, and four antibodies showed reduced immunostaining. In particular, excellent immunostaining results were obtained with a monoclonal antibody to vimentin as well as several different keratin antibodies on routine formalin-fixed tissue sections after pre-treatment of the slides with this method. These results showed that after antigen retrieval: (a) enzyme predigestion of tissues could be omitted; (b) incubation times of primary antibodies could be significantly reduced, or dilutions of primary antibodies could be increased; (c) adequate staining could be achieved in long-term formalin-fixed tissues that failed to stain by conventional methods; and (d) certain antibodies which were typically unreactive with formalin-fixed tissues gave excellent staining.

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