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

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.

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

2021 ◽  
Vol 4 (3) ◽  
pp. 47
Author(s):  
Yunguang Sun ◽  
Linna Ge ◽  
Sameer S. Udhane ◽  
John F. Langenheim ◽  
Mary J. Rau ◽  
...  
Keyword(s):  
Nucleocapsid Protein ◽  
Polyclonal Antibodies ◽  
Specific Detection ◽  
Viral Reservoirs ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Life Threatening ◽  
Log Linear ◽  
And Control ◽  
Formalin Fixed

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.

scholarly journals Immunofluorescence-guided segmentation of three-dimensional features in micro-computed tomography datasets of human lung tissue

2021 ◽  
Vol 8 (11) ◽  
Author(s):  
Matthew J. Lawson ◽  
Orestis L. Katsamenis ◽  
David Chatelet ◽  
Aiman Alzetani ◽  
Oliver Larkin ◽  
...  
Keyword(s):  
Computed Tomography ◽  
Lung Tissue ◽  
Human Lung ◽  
Three Dimensional ◽  
Micro Computed Tomography ◽  
3D Segmentation ◽  
Human Lung Tissue ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

Micro-computed tomography (µCT) provides non-destructive three-dimensional (3D) imaging of soft tissue microstructures. Specific features in µCT images can be identified using correlated two-dimensional (2D) histology images allowing manual segmentation. However, this is very time-consuming and requires specialist knowledge of the tissue and imaging modalities involved. Using a custom-designed µCT system optimized for imaging unstained formalin-fixed paraffin-embedded soft tissues, we imaged human lung tissue at isotropic voxel sizes less than 10 µm. Tissue sections were stained with haematoxylin and eosin or cytokeratin 18 in columnar airway epithelial cells using immunofluorescence (IF), as an exemplar of this workflow. Novel utilization of tissue autofluorescence allowed automatic alignment of 2D microscopy images to the 3D µCT data using scripted co-registration and automated image warping algorithms. Warped IF images, which were accurately aligned with the µCT datasets, allowed 3D segmentation of immunoreactive tissue microstructures in the human lung. Blood vessels were segmented semi-automatically using the co-registered µCT datasets. Correlating 2D IF and 3D µCT data enables accurate identification, localization and segmentation of features in fixed soft lung tissue. Our novel correlative imaging workflow provides faster and more automated 3D segmentation of µCT datasets. This is applicable to the huge range of formalin-fixed paraffin-embedded tissues held in biobanks and archives.

scholarly journals The Gene Expression of MMP-2 and TIMP-1 in Non-pathological Paratumoral and Autopsy Lung Tissues

2021 ◽  
Vol 4 (1) ◽  
pp. 61-65
Author(s):  
Elahe Esmaeili ◽  
◽  
Sara Ghaffarpour ◽  
Alireza Sadeghipour ◽  
Tooba Ghazanfari ◽  
...  
Keyword(s):  
Gene Expression ◽  
Lung Tissue ◽  
Matrix Metalloproteinase 2 ◽  
Control Group ◽  
Extracellular Matrix Remodeling ◽  
Matrix Remodeling ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed ◽  
Gene Expression Levels

Background: Finding a sample of healthy tissue is a critical challenge in research studies. Non-pathological Tissue adjacent to the tumor (NAT) specimens is usually used as the control in several studies. However, little is known about the similarity of NAT to healthy tissues. Here, we compared the expression of Matrix Metalloproteinase 2 (MMP-2) and its inhibitor, Tissue Inhibitors of MMP (TIMP)-1 as extracellular matrix remodeling factors in NAT and autopsy lung tissue. Materials and Methods: RNA of 7 NAT and 6 Formalin-Fixed Paraffin-Embedded (FFPE) lung autopsies from healthy people as the control group was extracted, and cDNA was synthesized. The gene expression levels of MMP-2 and TIMP-1 were evaluated by real-time PCR. Results: There were no significant differences in the expression of MMP-2, TIMP-1, or their ratio between the two groups. Conclusion: The results showed that NAT could be used as healthy controls in lung tissue studies for MMP-2 and TIMP-1.

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

1992 ◽  
Vol 4 (4) ◽  
pp. 393-399 ◽  
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.

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

2000 ◽  
Vol 12 (3) ◽  
pp. 224-232 ◽  
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.

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

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.

Rapid Polymerase Chain Reaction–Based Confirmation of Cat Scratch Disease and Bartonella henselae Infection

2003 ◽  
Vol 127 (6) ◽  
pp. 706-710
Author(s):  
Ben Margolis ◽  
Isinsu Kuzu ◽  
Marille Herrmann ◽  
Michele D. Raible ◽  
Eric Hsi ◽  
...  
Keyword(s):  
Bartonella Henselae ◽  
Pcr Amplification ◽  
Culture Collection ◽  
Tissue Sections ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Polymerase Chain ◽  
Negative Controls ◽  
Seminested Pcr ◽  
Formalin Fixed

Abstract Context.—Cat scratch disease (CSD) commonly occurs secondary to Bartonella henselae infection, and the diagnosis has traditionally been made by microscopic findings, the identification of organisms by cytochemistry, and clinical history. However, cytochemical analysis tends to be very difficult to interpret, and histology alone may be insufficient to establish a definitive diagnosis of CSD. Objective.—To demonstrate the presence of B henselae in tissue suspected of involvement by CSD, using a novel polymerase chain reaction (PCR) assay. Design.—Isolates of B henselae (American Tissue Culture Collection 49793) and Afipia felis (American Tissue Culture Collection 49714) were cultured on blood agar and buffered charcoal yeast extract agar, respectively. DNA was isolated from these organisms and from formalin-fixed, paraffin-embedded tissue sections with involvement by CSD (8 patients). Negative controls included water, human placental tissue, and lymph node specimens from 6 patients with reactive lymphoid hyperplasia and from 2 patients with granulomatous lymphadenitis. A primer complementary to B henselae citrate synthase gltA gene sequence was designed to perform a seminested PCR amplification. For restriction fragment length polymorphism analysis, PCR products were digested by TaqI restriction enzyme and analyzed by gel electrophoresis. Results.—Seminested PCR analysis of the cultured isolates of B henselae, but not of A felis, showed specific amplification. However, nonnested PCR did not provide consistently positive results in tissue sections with CSD. Therefore, we used a seminested PCR, which revealed positivity in all of the cases with clinicopathologic diagnoses of CSD. None of the negative controls showed positivity. Restriction enzyme provided confirmation of the specific PCR amplification of the B henselae sequence. Conclusions.—Since the amplification product has a low molecular size (<200 base pairs), this assay is useful for detection of B henselae in formalin-fixed, paraffin-embedded tissues. The seminested PCR protocol described here can be used for rapid and reliable confirmation of B henselae in samples that are histologically suggestive of CSD.

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