scholarly journals Influence of prolonged formalin fixation of tissue samples on the sensitivity of chromogenic in situ hybridization

2011 ◽  
Vol 23 (6) ◽  
pp. 1212-1216 ◽  
Author(s):  
Meike M. Mostegl ◽  
Barbara Richter ◽  
Nora Dinhopl ◽  
Herbert Weissenböck
Keyword(s):  
In Situ Hybridization ◽  
Nucleic Acid ◽  
Signal Intensity ◽  
Formalin Fixation ◽  
Proteinase K ◽  
Cross Linking ◽  
Fixation Time ◽  
Tissue Samples ◽  
Chromogenic In Situ Hybridization

Chromogenic in situ hybridization (ISH) is a commonly used tool in diagnostic pathology to detect pathogens in formalin-fixed, paraffin-embedded (FFPE) tissue sections. Prolonged formalin fixation time was identified to be a limiting factor for the successful detection of nucleic acid from different pathogens, most probably due to the cross-linking activity of formalin between RNA, DNA, and proteins. Therefore, in the current study, the influence of formalin fixation time on ISH signal intensity of 2 viral ( Porcine circovirus-2 [PCV-2] and Porcine respiratory and reproductive virus [PRRSV]) and 2 protozoal agents ( Cryptosporidium serpentis and Tritrichomonas sp.) was evaluated. Tissue samples were fixed in 7% neutral buffered formaldehyde solution, and at defined intervals, pieces were embedded in paraffin wax and subjected to pathogen-specific ISH. For all 4 pathogens, the signal intensity remained comparable with the starting ISH signal for different periods of fixation (PCV-2: 6 weeks, PRRSV: 23 weeks, C. serpentis: 55 weeks, Tritrichomonas sp.: 53 weeks). Thereafter, the signal started to decline until loss of nucleic acid detection. The influence of increased proteinase K concentrations for inverting the formalin-induced cross-linking activity was examined compared with the standard protocol. With all 4 infectious agents, a 4-fold proteinase K concentration restored the ISH signals to a level comparable with 1 day of fixation. In conclusion, the influence of prolonged formalin fixation on the intensity of detected ISH signal highly depends on the analyzed infectious agent and the pretreatment protocol.

scholarly journals A novel approach for microRNA in situ hybridization using locked nucleic acid probes

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Isabella W. Paulsen ◽  
Michael Bzorek ◽  
Jesper Olsen ◽  
Birgitte Grum-Schwensen ◽  
Jesper T. Troelsen ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Nucleic Acid ◽  
High Sensitivity ◽  
Staining Intensity ◽  
Locked Nucleic Acid ◽  
Proteinase K ◽  
Fixation Time ◽  
Target Tissue ◽  
Messenger Ribonucleic Acid

AbstractIdentification of target tissue microRNAs (miR) using in situ hybridization (ISH), with digoxigenin-labeled locked nucleic acid (LNA) probes, is influenced by preanalytic parameters. To determine the best retrieval method for common microRNAs, a multiblock composed of paraffin-embedded tonsil, cervix, placenta, and hyperplastic prostate tissue were included. Tissue were fixed in 10% formalin in a range of 5–144 hours (h). Cut sections (5 μm) from the multiblock were subjected to combinations of pretreatment procedures: variable periods of proteinase K (PK) digestion or Heat-induced microRNA Retrieval (HmiRR) using target retrieval solution (TRS) pH 6.1 or 9, with or without enzymatic treatment (pepsin). Results for the overall categories: TRS pH 9 versus PK; p = 2.9e−23, TRS pH 9 versus TRS pH 6.1; p = 1.1e−14, TRS pH 6.1 versus PK; p = 2.9e−03. A long fixation time, resulted in the best microRNA preservation and staining intensity (long vs. short: p = 3.5e−47, long vs. moderate: p = 1.6e−44, moderate vs. short: p = 4.3e−16), was enhanced using HmiRR TRS pH 9 with or without pepsin providing high sensitivity and specificity. These observations conflict with other ISH techniques (e.g., messenger ribonucleic acid), which typically require shorter fixation periods, and therefore, further studies are warranted.

scholarly journals Nonspecific binding of nucleic acid probes to Paneth cells in the gastrointestinal tract with in situ hybridization.

1992 ◽  
Vol 40 (10) ◽  
pp. 1613-1618 ◽  
Author(s):  
K L Garrett ◽  
M D Grounds ◽  
M W Beilharz
Keyword(s):  
In Situ Hybridization ◽  
Gastrointestinal Tract ◽  
Nucleic Acid ◽  
Paneth Cells ◽  
Proteinase K ◽  
Hybridization Technique ◽  
Nonspecific Binding ◽  
Nucleic Acid Probes ◽  
Hybridization Data

Nonspecific binding of a number of unrelated nucleic acid probes to cells in the crypts of Lieberkuhn was observed in the small intestine of mice with the in situ hybridization technique. Hybridization signal was localized to cells which, by virtue of their histological position, represented Paneth cells. This signal could not be removed by RNAse, DNAse, or proteinase K treatment, and was not removed after high-stringency washing conditions. This report indicates that caution must be exercised in the interpretation of in situ hybridization data when looking for nucleic acid sequences in the gastrointestinal tract.

scholarly journals Avian haemosporidian parasites of accipitriform raptors

2022 ◽  
Vol 21 (1) ◽  
Author(s):  
Josef Harl ◽  
Tanja Himmel ◽  
Gediminas Valkiūnas ◽  
Mikas Ilgūnas ◽  
Nora Nedorost ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Parasite Species ◽  
Sequence Data ◽  
Molecular Genetic ◽  
Tissue Samples ◽  
Host Group ◽  
Network Analyses ◽  
Haemosporidian Parasites ◽  
Chromogenic In Situ Hybridization

Abstract Background The order Accipitriformes comprises the largest group of birds of prey with 260 species in four families. So far, 21 haemosporidian parasite species have been described from or reported to occur in accipitriform birds. Only five of these parasite species have been characterized molecular genetically. The first part of this study involved molecular genetic screening of accipitriform raptors from Austria and Bosnia-Herzegovina and the first chromogenic in situ hybridization approach targeting parasites in this host group. The aim of the second part of this study was to summarize the CytB sequence data of haemosporidian parasites from accipitriform raptors and to visualize the geographic and host distribution of the lineages. Methods Blood and tissue samples of 183 accipitriform raptors from Austria and Bosnia-Herzegovina were screened for Plasmodium, Haemoproteus and Leucocytozoon parasites by nested PCR, and tissue samples of 23 PCR-positive birds were subjected to chromogenic in situ hybridization using genus-specific probes targeting the parasites’ 18S rRNAs. All published CytB sequence data from accipitriform raptors were analysed, phylogenetic trees were calculated, and DNA haplotype network analyses were performed with sequences from clades featuring multiple lineages detected in this host group. Results Of the 183 raptors from Austria and Bosnia-Herzegovina screened by PCR and sequencing, 80 individuals (44%) were infected with haemosporidian parasites. Among the 39 CytB lineages detected, 18 were found for the first time in the present study. The chromogenic in situ hybridization revealed exo-erythrocytic tissue stages of Leucocytozoon parasites belonging to the Leucocytozoon toddi species group in the kidneys of 14 infected birds. The total number of CytB lineages recorded in accipitriform birds worldwide was 57 for Leucocytozoon, 25 for Plasmodium, and 21 for Haemoproteus. Conclusion The analysis of the DNA haplotype networks allowed identifying numerous distinct groups of lineages, which have not yet been linked to morphospecies, and many of them likely belong to yet undescribed parasite species. Tissue stages of Leucocytozoon parasites developing in accipitriform raptors were discovered and described. The majority of Leucocytozoon and Haemoproteus lineages are specific to this host group, but most Plasmodium lineages were found in birds of other orders. This might indicate local transmission from birds kept at the same facilities (raptor rescue centres and zoos), likely resulting in abortive infections. To clarify the taxonomic and systematic problems, combined morphological and molecular genetic analyses on a wider range of accipitriform host species are needed.

A PILOT STUDY OF HER-2/NEU GENE AMPLIFICATION ASSESSED BY FLUORESCENCE IN SITU HYBRIDIZATION (FISH) IN GASTRIC CANCER

2014 ◽  
pp. 15-20
Author(s):  
Van Huy Tran ◽  
Thi Minh Thi Ha ◽  
Trung Nghia Van ◽  
Viet Nhan Nguyen ◽  
Phan Tuong Quynh Le ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Cancer Patients ◽  
Gene Amplification ◽  
Cancer Tissue ◽  
Tissue Samples ◽  
Her 2 ◽  
Gastric Cancer Patients

Background: HER-2/neu is a predictive biomarker for treatment of gastric cancer using trastuzumab in combination with chemotherapy. This study aimed to evaluate the status of HER-2/neu gene amplification using fluorescence in situ hybridization (FISH) in gastric cancer. Patients and methods: thirty six gastric cancer patients were assessed HER-2/neu gene amplification by FISH using PathVysionTM HER-2 DNA Probe kit (including HER-2/neu probe and CEP-17 probe) with biopsy and surgical specimens. Results: The HER-2/neu gene amplification was observed in three cases (8.3%), the HER-2/neu gene amplification rate in Lauren’s intestinal-type and diffuse-type were 11.8% and 5.2%, respectively. Conclusion: We applied successfully FISH technique with gastric cancer tissue samples. This technique could be performed as routine test in gastric cancer in order to select patients that benefit from trastuzumab in combination with chemotherapy.

scholarly journals Locked Nucleic Acid In situ Hybridization Analysis of miR-21 Expression during Colorectal Cancer Development

2009 ◽  
Vol 15 (12) ◽  
pp. 4009-4016 ◽  
Author(s):  
Nobutake Yamamichi ◽  
Ryoichi Shimomura ◽  
Ken-ichi Inada ◽  
Kouhei Sakurai ◽  
Takeshi Haraguchi ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
In Situ Hybridization ◽  
Nucleic Acid ◽  
Hybridization Analysis ◽  
Locked Nucleic Acid ◽  
Cancer Development
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