Journal of Cytology & Histology

Triple-Labeling Whole-Mount In Situ Hybridization Method for Analysis of Overlapping Gene Expression in Brain Tissue with High Level of Autofluorescence

Abstract

Jie Chen, Cindy Laramore and Michael Shifman

Background: Detection of multiple co-localized mRNAs by conventional chromogenic in situ hybridization is difficult because the first reaction product can obscure subsequent ones. Multi-color fluorescent in situ hybridization (FISH) offers simultaneous detection of multiple mRNAs but has low sensitivity and in cells of the central nervous system (CNS), is hampered by high background autofluorescence.
New method: Our method of sequential triple colorimetric in situ hybridization in whole-mounted lamprey brain, which has high background autofluorescence, uses three Alkaline Phosphatase (AP) substrates - Fast Red, 2-(4-iodophenyl)-5-(4-nitrophenyl)-3-phenyltetrazolium chloride (INT) /5-bromo-4-chloro-3-indolyl-phosphate (BCIP) and nitro blue tetrazolium (NBT) /BCIP, to develop red, yellow-brown and purple-blue signals sequentially. To achieve sequential colorimetric in situ hybridization, we selected AP substrates that produce ethanol-soluble reaction products, such as Fast Red and INT/BCIP. The most important step is to wash away the current color before the next color is developed.
Results: Using this sequential triple colorimetric in situ hybridization method, we showed that the netrin receptors deleted in colorectal cancer (DCC) and uncoordinated 5 (UNC-5), as well as the repulsive guidance molecule (RGM) receptor Neogenin, are all co-expressed in some reticulospinal neurons of the larval lamprey brainstem. The sensitivity and specificity of the fluorescence method in whole-mounted lamprey brain, which has a high degree of autofluorescence, were greater than those of a commercial colorimetric method, the Tyramide Signal Amplification (TSA) Fluorescence Kit.
Conclusions: The non-fluorescent chromogenic method is a simple, sensitive and reliable alternative to FISH on whole-mounted tissue with strong endogenous autofluorescence. And this sequential non-fluorescent chromogenic method allows detection of three overlapping or co-localized mRNA targets without loss of sensitivity in the second and third rounds of detection, and without masking of lighter signals by stronger ones.