Combined Radioactive and Nonradioactive Double In Situ Hybridization (DISH)
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Springer Science+Business Media New York 2015. This chapter describes in detail a reliable method for dual in situ hybridization (ISH) that has been validated in numerous studies identifying co-localization of high and low abundant target mRNAs in brain tissues. ISH is widely used to study the spatial distribution of a target mRNA with high anatomical resolution to localize the gene transcript in small areas and even in single cells. Both radioactive- and nonradioactive- labeled cRNA probes can be used for the detection of the complementary mRNA with high specificity and sensitivity. In order to localize two gene transcripts in the same tissue and establish co-expression which is especially valuable for determining mRNA expression in specific cell populations, these two approaches are combined to double in situ hybridization (DISH). Here we describe a protocol for the simultaneous use of isotopic with non-isotopic ISH in frozen brain sections where one probe is tagged with 35S-UTP and the other with digoxigenin (Dig)- UTP, respectively. In this protocol, Dig- labeled hybrids appear as purple cytoplasmic staining following detection with an alkaline-phosphatase- linked anti-dig antibody; S35-radioactive hybrids appear as silver grains after processing through conventional autoradiographic emulsion. This is a long and delicate procedure that results in a permanent record of target gene expression which can be analyzed even years later. We optimized this protocol for co- expression of nicotinic acetylcholine receptor (nAChR) subunits in glutamic acid decarboxylase (GAD67) mRNA positive neurons but it can be used to detect other mRNA targets with a wide range of gene expression intensity from high to low abundance and in different tissues.
