FISH on Histological Sections

Solovei, Irina; Grasser, Florian; Lanctôt, Christian

doi:10.1101/pdb.prot4729

Cited by 14 publications

(11 citation statements)

References 4 publications

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“…FISH was performed on sections as described elsewhere [99]. Briefly, sections were permeabilized in 0.5% Triton X-100 in PBS and then genomic DNA was unmasked by 9 cycles of incubation at 90°C using a microwave and cooling for 2 min in 10 mM sodium citrate buffer (pH 6.0).…”

Section: Methodsmentioning

confidence: 99%

Nipbl and Mediator Cooperatively Regulate Gene Expression to Control Limb Development

et al. 2014

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Haploinsufficiency for Nipbl, a cohesin loading protein, causes Cornelia de Lange Syndrome (CdLS), the most common “cohesinopathy”. It has been proposed that the effects of Nipbl-haploinsufficiency result from disruption of long-range communication between DNA elements. Here we use zebrafish and mouse models of CdLS to examine how transcriptional changes caused by Nipbl deficiency give rise to limb defects, a common condition in individuals with CdLS. In the zebrafish pectoral fin (forelimb), knockdown of Nipbl expression led to size reductions and patterning defects that were preceded by dysregulated expression of key early limb development genes, including fgfs, shha, hand2 and multiple hox genes. In limb buds of Nipbl-haploinsufficient mice, transcriptome analysis revealed many similar gene expression changes, as well as altered expression of additional classes of genes that play roles in limb development. In both species, the pattern of dysregulation of hox-gene expression depended on genomic location within the Hox clusters. In view of studies suggesting that Nipbl colocalizes with the mediator complex, which facilitates enhancer-promoter communication, we also examined zebrafish deficient for the Med12 Mediator subunit, and found they resembled Nipbl-deficient fish in both morphology and gene expression. Moreover, combined partial reduction of both Nipbl and Med12 had a strongly synergistic effect, consistent with both molecules acting in a common pathway. In addition, three-dimensional fluorescent in situ hybridization revealed that Nipbl and Med12 are required to bring regions containing long-range enhancers into close proximity with the zebrafish hoxda cluster. These data demonstrate a crucial role for Nipbl in limb development, and support the view that its actions on multiple gene pathways result from its influence, together with Mediator, on regulation of long-range chromosomal interactions.

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Section: Methodsmentioning

confidence: 99%

Nipbl and Mediator Cooperatively Regulate Gene Expression to Control Limb Development

et al. 2014

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“…Briefly, cosmids 14, 28 and 56 [104] comprising a total of ∼90 kb of HSV-1 genome (see Figure 1A) were labeled by Nick translation (Roche Diagnostic) with dCTP-Cy3 (GE Healthcare), and stored in 100% formamide (Sigma-Aldrich). The DNA-FISH procedure was adapted from Solovei et al [105].…”

Section: Methodsmentioning

confidence: 99%

HSV-1 Genome Subnuclear Positioning and Associations with Host-Cell PML-NBs and Centromeres Regulate LAT Locus Transcription during Latency in Neurons

et al. 2012

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Major human pathologies are caused by nuclear replicative viruses establishing life-long latent infection in their host. During latency the genomes of these viruses are intimately interacting with the cell nucleus environment. A hallmark of herpes simplex virus type 1 (HSV-1) latency establishment is the shutdown of lytic genes expression and the concomitant induction of the latency associated (LAT) transcripts. Although the setting up and the maintenance of the latent genetic program is most likely dependent on a subtle interplay between viral and nuclear factors, this remains uninvestigated. Combining the use of in situ fluorescent-based approaches and high-resolution microscopic analysis, we show that HSV-1 genomes adopt specific nuclear patterns in sensory neurons of latently infected mice (28 days post-inoculation, d.p.i.). Latent HSV-1 genomes display two major patterns, called “Single” and “Multiple”, which associate with centromeres, and with promyelocytic leukemia nuclear bodies (PML-NBs) as viral DNA-containing PML-NBs (DCP-NBs). 3D-image reconstruction of DCP-NBs shows that PML forms a shell around viral genomes and associated Daxx and ATRX, two PML partners within PML-NBs. During latency establishment (6 d.p.i.), infected mouse TGs display, at the level of the whole TG and in individual cells, a substantial increase of PML amount consistent with the interferon-mediated antiviral role of PML. “Single” and “Multiple” patterns are reminiscent of low and high-viral genome copy-containing neurons. We show that LAT expression is significantly favored within the “Multiple” pattern, which underlines a heterogeneity of LAT expression dependent on the viral genome copy number, pattern acquisition, and association with nuclear domains. Infection of PML-knockout mice demonstrates that PML/PML-NBs are involved in virus nuclear pattern acquisition, and negatively regulate the expression of the LAT. This study demonstrates that nuclear domains including PML-NBs and centromeres are functionally involved in the control of HSV-1 latency, and represent a key level of host/virus interaction.

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“…For general background and description of conventional manipulation on ISH, IF and FISH, we suggest the following available literature 23 .…”

Section: Protocolmentioning

confidence: 99%

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent <i>In situ</i> Hybridization Combined with Immunostaining

Catez¹,

Rousseau²,

Labétoulle³

et al. 2014

JoVE

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Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge in the field of virology; in particular for nuclear-replicating persistent DNA viruses that involve animal models for their study. Herpes simplex virus type 1 (HSV-1) establishes a life-long latent infection in peripheral neurons. Latent virus serves as reservoir, from which it reactivates and induces a new herpetic episode. The cell biology of HSV-1 latency remains poorly understood, in part due to the lack of methods to detect HSV-1 genomes in situ in animal models. We describe a DNA-fluorescent in situ hybridization (FISH) approach efficiently detecting low-copy viral genomes within sections of neuronal tissues from infected animal models. The method relies on heat-based antigen unmasking, and directly labeled home-made DNA probes, or commercially available probes. We developed a triple staining approach, combining DNA-FISH with RNA-FISH and immunofluorescence, using peroxidase based signal amplification to accommodate each staining requirement. A major improvement is the ability to obtain, within 10 µm tissue sections, low-background signals that can be imaged at high resolution by confocal microscopy and wide-field conventional epifluorescence. Additionally, the triple staining worked with a wide range of antibodies directed against cellular and viral proteins. The complete protocol takes 2.5 days to accommodate antibody and probe penetration within the tissue.

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FISH on Histological Sections

Cited by 14 publications

References 4 publications

Nipbl and Mediator Cooperatively Regulate Gene Expression to Control Limb Development

Nipbl and Mediator Cooperatively Regulate Gene Expression to Control Limb Development

HSV-1 Genome Subnuclear Positioning and Associations with Host-Cell PML-NBs and Centromeres Regulate LAT Locus Transcription during Latency in Neurons

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent <i>In situ</i> Hybridization Combined with Immunostaining

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