An efficient in situ hybridization protocol for multiple tissue sections and probes on miniaturized slides.
To facilitate detection of gene activity in tissue sections we combined common protocols of in situ hybridization on tissue sections (TSISH) with the technique of whole-mount in situ hybridization (WMISH). Miniature glass slides for mounting tissue sections were cut from regular microscope slides and handled for in situ hybridization in laboratory-made 2-ml containers (baskets) similar to those originally used for WMISH on Drosophila embryos. A salient point of the method is the use of airtight reaction vessels placed in a dry thermostat for critical hybridization steps as this facilitates reproducible and stringent hybridization conditions which are difficult to achieve on tissue sections otherwise. The practicability of the method is illustrated on consecutive serial frozen sections of murine neonatal cerebellum hybridized for math1 and neuroD, two developmentally regulated genes with distinct expression patterns. For both genes excellent spatial resolution and a highly dynamic range of signal intensity was obtained. The approach enables simple processing of multiple probes, allows the efficient and economic use of small tissue samples and is amenable to automation.
Weisheit
Gunnar
G
Anatomisches Institut, Anatomie und Zellbiologie Rheinische Friedrich-Wilhelms-Universität, Nussallee 10, 53115 Bonn, Germany.
Mertz
Diana
D
Schilling
Karl
K
Viebahn
Christoph
C
D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins; D002531:Cerebellum / Q000254:growth & development; D016616:Cryoultramicrotomy; D015342:DNA Probes; D018507:Gene Expression Regulation, Developmental; D017403:In Situ Hybridization / Q000379:methods*; D051379:Mice; D009419:Nerve Tissue Proteins / Q000235:genetics; D016707:Tissue Fixation; D014157:Transcription Factors / Q000235:genetics
D051792:Basic Helix-Loop-Helix Proteins; D015342:DNA Probes; D009419:Nerve Tissue Proteins; D014157:Transcription Factors; C500943:Atoh1 protein, mouse; C093564:Neurogenic differentiation factor 1