PMID- 22479470 OWN - NLM STAT- MEDLINE VI - 7 IP - 3 TI - A rapid and highly sensitive method of non radioactive colorimetric in situ hybridization for the detection of mRNA on tissue sections. PG - e33898 LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't PL - United States TA - Plos One JT - PloS one JID - 101285081 IS - 1932-6203 (Electronic) LID - 10.1371/journal.pone.0033898 [doi] FAU - Stylianopoulou, Electra AU - Stylianopoulou E AD - Department of Molecular Biology and Genetics, Democritus University of Thrace, Alexandroupolis, Greece. FAU - Lykidis, Dimitrios AU - Lykidis D FAU - Ypsilantis, Petros AU - Ypsilantis P FAU - Simopoulos, Constantinos AU - Simopoulos C FAU - Skavdis, George AU - Skavdis G FAU - Grigoriou, Maria AU - Grigoriou M IS - 1932-6203 (Linking) RN - 0 (RNA, Messenger) SB - IM MH - Animals MH - Colorimetry MH - Cryoultramicrotomy MH - Embryo, Mammalian/*cytology/*metabolism MH - Gene Expression MH - Histocytochemistry MH - In Situ Hybridization/*methods MH - Mice MH - RNA, Messenger/*analysis MH - Sensitivity and Specificity PMC - PMC3316699 DCOM- 20121119 LR - 20211021 DP - 2012 DEP - 20120330 AB - 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. METHODOLOGY/PRINCIPAL FINDINGS: 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/SIGNIFICANCE: 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.