scholarly journals Development of immunohistochemistry and in situ hybridisation for the detection of SARS-CoV and SARS-CoV-2 in formalin-fixed paraffin-embedded specimens

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Fabian Z. X. Lean ◽  
Mart M. Lamers ◽  
Samuel P. Smith ◽  
Rebecca Shipley ◽  
Debby Schipper ◽  
...  
Keyword(s):  
Animal Models ◽  
In Situ Hybridisation ◽  
Virus Antigen ◽  
Control Material ◽  
Live Virus ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Infected Cells ◽  
Formalin Fixed

AbstractThe rapid emergence of SARS-CoV-2, the causative agent of COVID-19, and its dissemination globally has caused an unprecedented strain on public health. Animal models are urgently being developed for SARS-CoV-2 to aid rational design of vaccines and therapeutics. Immunohistochemistry and in situ hybridisation techniques that facilitate reliable and reproducible detection of SARS-CoV and SARS-CoV-2 viral products in formalin-fixed paraffin-embedded (FFPE) specimens would be of great utility. A selection of commercial antibodies generated against SARS-CoV spike protein and nucleoprotein, double stranded RNA, and RNA probe for spike genes were evaluated for the ability to detect FFPE infected cells. We also tested both heat- and enzymatic-mediated virus antigen retrieval methods to determine the optimal virus antigen recovery as well as identifying alternative retrieval methods to enable flexibility of IHC methods. In addition to using native virus infected cells as positive control material, the evaluation of non-infected cells expressing coronavirus (SARS, MERS) spike as a biosecure alternative to assays involving live virus was undertaken. Optimized protocols were successfully applied to experimental animal-derived tissues. The diverse techniques for virus detection and control material generation demonstrated in this study can be applied to investigations of coronavirus pathogenesis and therapeutic research in animal models.

scholarly journals Detection protocols for biotinylated probes: optimization using multistep techniques.

1992 ◽  
Vol 40 (4) ◽  
pp. 569-574 ◽  
Author(s):  
S McQuaid ◽  
G M Allan
Keyword(s):  
Nucleic Acid ◽  
Measles Virus ◽  
Single Step ◽  
Step Detection ◽  
Formalin Fixed Paraffin ◽  
Wide Range ◽  
Formalin Fixed Paraffin Embedded ◽  
Infected Cells ◽  
Formalin Fixed

Recent studies using biotinylated in situ hybridization (ISH) have utilized a wide range of detection protocols for the biotinylated hybrids, leading to conflicting reports in the literature regarding sensitivity. In this study we compared 11 different detection protocols for biotinylated ISH using a measles virus-specific RNA probe on formalin-fixed, paraffin-embedded central nervous system tissue infected with measles virus. Maximum sensitivity was achieved with five-step detection protocols incorporating the use of a monoclonal antibody to biotin. Single-step detection protocols were found to be insensitive, as shown by their failure to detect viral nucleic acid in infected white-matter cells. Only by increasing the number of steps in the detection protocols were these infected cells demonstrable. Unless pre-hybridization, hybridization, and detection protocols are optimized, the results obtained in pathogenicity studies using ISH could be misinterpreted, leading to false conclusions about nucleic acid distribution. This also applies to the ever-increasing use of ISH for diagnostic purposes.

Evaluation of two commercialised in situ hybridisation assays for detecting HPV-DNA in formalin-fixed, paraffin-embedded tissue

2010 ◽  
Vol 284 (4) ◽  
pp. 999-1005 ◽  
Author(s):  
Maresa Montag ◽  
Thomas J. F. Blankenstein ◽  
Naim Shabani ◽  
Ansgar Brüning ◽  
Ioannis Mylonas
Keyword(s):  
In Situ Hybridisation ◽  
Hpv Dna ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

Canine Hepatitis Associated with Intrahepatic Bacteria in Three Dogs

2018 ◽  
Vol 54 (1) ◽  
pp. 65-70 ◽  
Author(s):  
Joon Im ◽  
Derek P. Burney ◽  
Sean P. McDonough ◽  
Brigid Nicholson ◽  
Adam Eatroff ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Case Report ◽  
Fluorescence In Situ Hybridization ◽  
Antimicrobial Therapy ◽  
Granulomatous Inflammation ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Hematoxylin And Eosin ◽  
Formalin Fixed

ABSTRACT This case report describes the detection of intrahepatic bacteria in formalin-fixed paraffin-embedded histopathological sections from three dogs with neutrophilic, pyogranulomatous, or lymphoplasmacytic hepatitis and cholangiohepatitis. In each of these cases, eubacterial fluorescence in situ hybridization enabled colocalization of intrahepatic bacteria with neutrophilic and granulomatous inflammation in samples that were negative for bacteria when evaluated by routine hematoxylin and eosin histopathology augmented with histochemical stains. Positive responses to antimicrobial therapy were observed in of 2 out of 2 patients that were treated with antimicrobials. These findings suggest that eubacterial fluorescence in situ hybridization analysis of formalin-fixed paraffin-embedded histopathological sections is more sensitive than conventional histochemical stains for the diagnosis of bacteria-associated canine hepatitis.

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