Signal intensities of radiolabeled cRNA probes used alone or in combination with non-isotopic in situ hybridization histochemistry

Abstract

This study addressed the question of whether radioactive hybridization signal intensities are reduced in combined isotopic and non-isotopic double in situ hybridization (DISH) compared with those in single in situ hybridization (ISH). Non-isotopic digoxigenin (Dig)-labeled hybrids were detected using an alkaline phosphatase (AP) enzymatic reaction which results in nitroblue tetrazolium chloride (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP)-salt precipitation that could shield S35-radiation from penetrating to the surface. Sections were plastic coated of with 2% parlodion to prevent a chemical reaction between AP and developer during processing of the photosensitive emulsion, which could further reduce radioactive hybridization signal detection by autoradiography. We used DISH with a hybridization cocktail of radioactive S35- and Dig-labeled GAD67 cRNA probes. In order to avoid competition for the same complementary sequence, the probes were directed towards different sequences of the glutamic acid decarboxylase (GAD67) mRNA, resulting in co-detection of isotopic and non-isotopic hybrids in close to 100% of GAD67 positive cells. Quantitation of autoradiograms showed that there was no reduction of autoradiographic signal intensity from S35-labeled hybrids in the presence of Dig-labeled hybrids. Plastic coating of single or dual hybridized sections did not reduce the radioactive signal intensity. When mRNAs for nicotinic acetylcholine receptor (nAChR) subunits were detected with subunit specific S35-labeled cRNA probes in GAD67 hippocampal interneurons the total numbers of nAChR subunit expressing cells remained the same in single or double hybridized sections even for low abundant mRNAs. Together, these results indicate that combined radioactive and non-radioactive DISH does not interfere with the detection of the radiation signal from the S35-labeled hybrids, and neither specificity nor sensitivity is compromised.

Figure 1

GAD67 mRNA hybridization patterns generated with dual in situ hybridization using Dig- and S³5-labeled GAD67 cRNA probes. The hybridization signals in hippocampus (A, A′, a), cingulated cortex (B, B′, b) and reticular thalamic nucleus (C, C′, c) are shown for Dig-GAD67 in lightfield (A, B, C), S³⁵-GAD67 in darkfield, and together in darkfield/lightfield (a, b, c). Frames a, b and c correspond to higher magnification of areas delineated in A′, B′ and C′. Scale bar = 1 mm in C′ applies to A-C′, and 100 μm in c applies to a–c.

Figure 2

A) Autoradiographic images of S³⁵-GAD67 hybridization patterns generated with single (left side of the sections) or dual (right side of the sections) in situ hybridization using a combination of S³⁵- and Dig-labeled GAD67 cRNA probes, before (left) and after 2% parlodion coating (right). B) Quantitation of the S³⁵-hybridization signal intensity in cingulate cortex (Cg), caudate putamen (CPu) and reticular thalamic nucleus (Rt) in single and dual in situ hybridization, before and after 2% parlodion coating. Error bars indicate S.E.M.. Scale bar = 1mm in A.

Figure 3

Darkfield photomicrographs of S³⁵-α4 nAChR subunit hybridization in the hippocampus using a S³⁵-labeled α4 nAChR subunit cRNA probe in single (A), and dual in situ hybridization in combination with a Dig-GAD67 cRNA probe (B). Frame a in A corresponds to higher magnification of the area delineated in panel a. Frames b and c correspond to the higher magnification of areas delineated in panel b and c. White arrow heads indicate cells positive for α4 hybridization; black arrow indicates Dig-GAD67 hybridization only; * indicates co-labeled cell positive for S³⁵-α4 and Dig-GAD67 hybridization. Scale bars: 1 mm in B applies to A and B; 200 μm in b applies to a and b; 100μm in c.

Figure 4

Quantitation of rat brain sections processed for dual and single ISH using S³⁵-labeled probes for nAChR subunits with or without Dig-GAD67 cRNA probe, respectively. The number of neurons exhibiting positive hybridization signal for α2, α3, α4, α5, α7, β2 and β4 nAChR subunit mRNAs were counted in the hippocampus in sections from single and double ISH. Error bars indicate S.E.M.