title: Detection of the genome and transcripts of a persistent DNA virus in neuronal tissues by fluorescent in situ hybridization combined with immunostaining.
abstract: 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.
authors: Virus and Centromere Team, Centre de Génétique et Physiologie Moléculaire et Cellulaire, CNRS UMR 5534.
authors: Frédéric
authors: F
authors: Catez
authors: Antoine
authors: A
authors: Rousseau
authors: Marc
authors: M
authors: Labetoulle
authors: Patrick
authors: P
authors: Lomonte
mesh_terms: D000818:Animals; D004279:DNA, Viral / Q000032:analysis / Q000235:genetics; D005260:Female; D005455:Fluorescent Antibody Technique / Q000379:methods*; D016679:Genome, Viral; D018259:Herpesvirus 1, Human / Q000235:genetics*; D007124:Immunoenzyme Techniques; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D051379:Mice; D008807:Mice, Inbred BALB C; D018613:Microscopy, Confocal; D009474:Neurons / Q000821:virology*; D012367:RNA, Viral / Q000032:analysis / Q000235:genetics
chemical_list: D004279:DNA, Viral; D012367:RNA, Viral