title: A rapid and highly sensitive method of non radioactive colorimetric in situ hybridization for the detection of mRNA on tissue sections.
abstract: BACKGROUND
  Non Radioactive colorimetric In Situ Hybridization (NoRISH) with hapten labeled probes has been widely used for the study of gene expression in development, homeostasis and disease. However, improvement in the sensitivity of the method is still needed to allow for the analysis of genes expressed at low levels.
  
  
  RESULTS
  A stable, non-toxic, zinc-based fixative was tested in NoRISH experiments on sections of mouse embryos using four probes (Lhx6, Lhx7, ncapg and ret) that have different spatial patterns and expression levels. We showed that Z7 can successfully replace paraformaldehyde used so far for tissue fixation in NoRISH; the morphology of the cryosections of Z7-fixed tissues was excellent, and the fixation time required for tissues sized 1 cm was 1 hr instead of 24 hr for paraformaldehyde. The hybridization signal on the sections of the Z7-treated embryos always appeared earlier than that of the PFA-fixed embryos. In addition, a 50-60% shorter detection time was observed in specimen of Z7-treated embryos, reducing significantly the time required to complete the method. Finally and most importantly, the strength of the hybridization signal on the sections of the Z7-treated embryos always compared favorably to that of the sections of PFA-fixed embryos; these data demonstrate a significant improvement of the sensitivity the method that allows for the analysis of mRNAs that are barely or not detected by the standard colorimetric NoRISH method.
  
  
  CONCLUSIONS
  Our NoRISH method provides excellent preservation of tissue morphology, is rapid, highly sensitive, and especially suitable to implement in the study of genes expressed at low levels and/or in sparse cells within a structure.
authors.affiliation: Department of Molecular Biology and Genetics, Democritus University of Thrace, Alexandroupolis, Greece.
authors.forename: Electra
authors.initials: E
authors.lastname: Stylianopoulou
authors.forename: Dimitrios
authors.initials: D
authors.lastname: Lykidis
authors.forename: Petros
authors.initials: P
authors.lastname: Ypsilantis
authors.forename: Constantinos
authors.initials: C
authors.lastname: Simopoulos
authors.forename: George
authors.initials: G
authors.lastname: Skavdis
authors.forename: Maria
authors.initials: M
authors.lastname: Grigoriou
mesh_terms: D000818:Animals; D003124:Colorimetry; D016616:Cryoultramicrotomy; D004622:Embryo, Mammalian / Q000166:cytology* / Q000378:metabolism*; D015870:Gene Expression; D006651:Histocytochemistry; D017403:In Situ Hybridization / Q000379:methods*; D051379:Mice; D012333:RNA, Messenger / Q000032:analysis*; D012680:Sensitivity and Specificity
chemical_list: D012333:RNA, Messenger