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Structural Methods in the Study of Development of the Lung
Published in Joan Gil, Models of Lung Disease, 2020
Paul Davies, Daphne deMello, Lynne M. Reid
A cell function expressed in a protein product can now be studied dynamically by determining when the controlling gene is transcribed (deMello et al., 1988). In situ hybridization is a powerful tool that allows posttranscriptional gene expression to be studied within cells in tissue. In situ hybridization (ISH) uses nicktranslated DNA or RNA probes. To make single-stranded DNA probes complementary to specific genes, plasmid DNA containing the gene is nicked, and repaired with radiolabeled nucleotides. The plasmid is then cut and denatured to form single-stranded fragments that include fragments complementary to the gene to be studies. For RNA probes, antisense probes are generated from plasmid vectors. The relevant cDNA fragment is inserted within these vectors in reverse orientation, so that promoter sequences able to transcribe this fragment will be positioned at the far end of the gene fragment, thus transcribing an opposite or antisense strand. In medium containing radiolabeled nucleotides, a specific RNA polymerase promotes the synthesis of labeled antisense RNA. This hybridizes with complementary RNA in fixed frozen or paraffin-wax-embedded tissue sections. Often, ISH and immunohistochemistry (IH) are performed on consecutive adjacent sections to correlate posttranscriptional and posttranslational gene expression.
Histological approaches
Published in Maxine Lintern, Laboratory Skills for Science and Medicine, 2018
In situ hybridisation is a system for detecting the presence of DNA and RNA in both histological and cytological preparations. Essentially as in Southern and/or northern blotting, the method relies on the fact that complementary nucleotide sequences will hybridise together. However, the main advantage of in situ hybridisation over these related techniques is that it allows for the specific cells containing the nucleic acids of interest to be visualised. Hence by using labelled nucleic acid sequences as probes complementary to the sequence of interest, both the existence and the position of the target can be verified.
Tissue and Molecular Diagnosis
Published in Professor Sir Norman Williams, Professor P. Ronan O’Connell, Professor Andrew W. McCaskie, Bailey & Love's Short Practice of Surgery, 2018
Professor Sir Norman Williams, Professor P. Ronan O’Connell, Professor Andrew W. McCaskie
In situ hybridisation (ISH) uses a labelled oligonucleotide probe targeted at a specific sequence of RNA or DNA. It can be performed on fixed or fresh tissue sections, allowing the presence or absence of a particular sequence and its location to be seen in situ. Autoradiography, fluorescence microscopy or immunohistochemistry can be used for visualisation. Chromogenic in situ hybridisation (CISH) combines ISH and immunohistochemistry for the detection of DNA regions and it is widely and routinely used in pathology laboratories for the detection of HER2 amplifications. Viral genomes, e.g. EBV (Figure16.28), CMV and human papillomavirus, can be detected using this approach. ISH plays an important role in the management of tumours (see below).
Strategies to improve the diagnosis and clinical treatment of dermatophyte infections
Published in Expert Review of Anti-infective Therapy, 2023
Nonetheless, it is not possible to distinguish fungal species using routine histopathological examinations. As most of these special dyes stain all fungi, the in situ hybridization method should be used if the fungal pathogen of the patient has not been determined by culture or molecular methods [23]. For in situ hybridization, different probes are used to detect the presence of specific fungal nucleic acids. During these tests, a thin section of tissue is placed on a slide, and is followed by direct hybridization. Similarly as in immunohistochemistry, the tissue can be frozen or paraffinized. Paraffin-embedded tissues require deparaffinization and rehydration before hybridization. Although not all fungal pathogens are found internally, DNA probes have been developed for some genera (e.g. Blastomyces, Coccidioides, Cryptococcus, Sporothrix, Pneumocystis, Candida, Fusarium, and Pseudallescheria) [24,25].
MicroRNA-122-5p ameliorates tubular injury in diabetic nephropathy via FIH-1/HIF-1α pathway
Published in Renal Failure, 2022
Li Cheng, Xinying Qiu, Liyu He, Li Liu
Fluorescence in situ hybridization (FISH) was performed according to the manufacturer's instructions. Briefly, kidneys were harvested from control and STZ-treated mice to prepare 4-micron paraffin section. The sections were treated with 20 μg/ml proteinase K for permeabilization, and then incubated with pre-hybridization solution at 78 °C for 1 h. Remove pre-hybridization solution and add digoxigenin-labeled mmu-miR-122-5p LNA probe over night at 37 °C. At the second day, after wash, bovine serum albumin (BSA) was added for blocking. Then the anti-digoxigenin-HRP was used at 37 °C for 1 h. CY3-TSA and DAPI assay were used to indicate the positive areas and cell nucleus respectively. The images were acquired from a fluorescence microscope and the representative figures were exhibited.
Different cellular mechanisms from low- and high-dose zinc oxide nanoparticles-induced heart tube malformation during embryogenesis
Published in Nanotoxicology, 2022
Mengwei Wang, Ping Zhang, Zeyu Li, Yu Yan, Xin Cheng, Guang Wang, Xuesong Yang
Whole-mount in situ hybridization was performed on chicken embryos according to the previously described standard protocol (Henrique et al. 1995). Total RNAs were isolated from HH10 chicken embryos. RNA antisense Wnt3a, FGF8, NKX2.5, GATA4, CSRP3 and MYL2 probes were obtained by reverse transcription polymerase chain reaction (RT-PCR) technique as previously described (Bales et al. 1993). Digoxigenin-labeled antisense probes were synthesized to specifically detect the expressions of Wnt3a, FGF8, NKX2.5, GATA4, CSRP3 and MYL2 at mRNA level. The primers used to generate the in situ hybridization probes are shown in Supplementary Table. 1. The whole-mount stained embryos were photographed and then frozen sections (thickness of 20 μm) were implemented for photography of the corresponding sections.