scholarly journals Non-isotopic in situ hybridization method for mitochondria in oncocytes.

1994 ◽  
Vol 42 (2) ◽  
pp. 273-276 ◽  
Author(s):  
V A Varma ◽  
C M Cerjan ◽  
K L Abbott ◽  
S B Hunter
Keyword(s):  
Mitochondrial Dna ◽  
In Situ Hybridization ◽  
Coding Region ◽  
Chain Reaction ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Polymerase Chain ◽  
Endogenous Biotin ◽  
Formalin Fixed

We used in situ hybridization to specifically identify mitochondria in a series of formalin-fixed, paraffin-embedded oncocytic lesions. Digoxigenin-labeled DNA probes were generated by the polymerase chain reaction (PCR), with primers designed to amplify a mitochondrion-specific 154 BP sequence within the ND4 coding region. Probes were hybridized with mitochondrial DNA under stringent conditions. Oncocytes were strongly and consistently stained, reflecting the high copy number of mitochondrial DNA within these cells. Because of the presence of endogenous biotin within mitochondria, digoxigenin is preferable to biotin as a label for detection of mitochondria.

scholarly journals Detection of Human Papillomavirus in Archival Tissues: Comparison of In Situ Hybridization and Polymerase Chain Reaction

1998 ◽  
Vol 46 (4) ◽  
pp. 535-540 ◽  
Author(s):  
Elizabeth R. Unger ◽  
Suzanne D. Vernon ◽  
Daisy R. Lee ◽  
Donna L. Miller ◽  
William C. Reeves
Keyword(s):  
Polymerase Chain Reaction ◽  
In Situ Hybridization ◽  
Human Papillomavirus ◽  
Chain Reaction ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Polymerase Chain ◽  
Epidemiology Studies ◽  
Assay Conditions

Formalin-fixed, paraffin-embedded tissues in pathology archives are an important resource for molecular epidemiology studies. Use of these tissues requires that assays be optimized to account for inevitable variations in tissue fixation and processing that occur in the performance of routine histology. We compared results of colorimetric in situ hybridization (ISH) to L1 consensus polymerase chain reaction (PCR) for detection and typing of human papillomavirus (HPV) in 180 blocks of archival tissues (up to 9 years in storage) from cervical cancer patients. Fifteen samples could not be amplified by PCR, but assays were concordant in 75.1% (124/165) of samples that could be analyzed by both methods. Similar numbers of ISH+/PCR- (23) and ISH-/PCR+ (18) cases were found. Eight of the 18 ISH-/PCR+ cases were attributable to PCR detection of HPV types not included in the ISH assay. This degree of concordance required individual optimization of assay conditions for each block. ISH and PCR assays for HPV yield complementary results, and both can be successfully applied to archival tissues.

scholarly journals Nested Polymerase Chain Reaction and In Situ Hybridization for Diagnosis of Canine Herpesvirus Infection in Puppies

1998 ◽  
Vol 35 (3) ◽  
pp. 209-217 ◽  
Author(s):  
C. Schulze ◽  
W. Baumgärtner
Keyword(s):  
Epithelial Cells ◽  
Chain Reaction ◽  
Viral Dna ◽  
Nested Polymerase Chain Reaction ◽  
Canine Herpesvirus ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Polymerase Chain ◽  
Formalin Fixed

The usefulness of two nucleic acid detection systems in suspected cases of spontaneous canine herpesvirus (CHV) infection in puppies was evaluated. Formalin-fixed, paraffin-embedded tissues from seven 1–3-week-old naturally infected puppies with lesions characteristic of CHV infection were investigated in a retrospective study. Nested polymerase chain reaction (PCR) and nonradioactive in situ hybridization (ISH) were used to detect nucleotide sequences of the CHV thymidine kinase (TK) gene. According to the original necropsy reports, CHV was isolated in four of the seven puppies using primary canine lung and/or kidney cells. In all seven puppies, gross and histologic lesions consisted of disseminated focal necroses and hemorrhages predominantly in kidneys, lung, liver, and spleen. In addition, few small amphophilic intranuclear inclusion bodies were detected by light microscopy mainly in epithelial cells of kidney, lung, and liver. ISH was performed with a 111-base-pair (bp) digoxigenin-labeled double-stranded DNA probe. Viral DNA was detected in the nuclei of cells near and within lesions. Various cell types, including bronchiolar and alveolar epithelial cells, hepatocytes, renal tubular epithelial cells, neurons, fibrocytes, cardiac myocytes, and endothelial cells, were positive for viral DNA. PCR amplification products of the expected length of 168 bp containing the expected cleavage site for the restriction enzyme EcoRI, derived from paraffin blocks containing lung, kidney, and liver tissues, were detected in all seven puppies. The specificity of the obtained amplicon was further confirmed by Southern blot analysis. ISH and PCR are both useful methods for diagnosing CHV infection in formalin-fixed, paraffin-embedded tissues and are highly specific and sensitive methods for further investigations of the pathogenesis of CHV-induced lesions.

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