Elsevier

Methods

Volume 52, Issue 4, December 2010, Pages 359-366
Methods

A quantitative in situ hybridization protocol for formalin-fixed paraffin-embedded archival post-mortem human brain tissue

https://doi.org/10.1016/j.ymeth.2010.07.004Get rights and content

Abstract

The use of radioactive in situ hybridization (ISH) to quantitatively determine low-to-moderate abundant mRNA expression in formalin-fixed, paraffin-embedded archival post-mortem human brain tissue is often limited by non-specific-deposits, visible as speckles. In the present study, optimal hybridization conditions were achieved for quantifying the mRNA expression of histidine decarboxylase (HDC) by a number of alterations in a routine protocol, which included (1) during purification of the oligo-probes, glycogen was omitted as a carrier for precipitation, (2) after precipitation, the labeled probe contained within the pellet was first dissolved in water instead of in hybridization buffer (HBF), (3) during hybridization, the dithiothreitol (DTT) concentration was increased from 200 to 800 mM in HBF, and (4) stringencies during hybridization and post-hybridization washes were increased by increasing the temperature. The effect of the adjustment was quantified on adjacent sections from 18 subjects (9 with Parkinson’s disease and 9 controls), by comparing the data from the standard and new protocol. The results showed that the improved protocol brought about significantly clearer background with higher signal-to-noise ratios (p = 0.001). We propose that this protocol is also applicable for detection of other lower-abundant genes in human brain tissue and probably in other tissues as well. In the present study, this is not only illustrated for HDC ISH, but also for corticotrophin-releasing hormone mRNA expression in the hypothalamic paraventricular nucleus.

Introduction

In situ hybridization (ISH) allows specific nucleic acid sequences to be detected in morphologically preserved tissue sections. This technique is widely used to detect the expression of specific genes at the mRNA level. When relative quantitative comparison between experimental groups is needed, radioactive ISH still holds the unique advantage of signal quantification. Although frozen tissue sections are often used for ISH, paraffin-embedded tissue sections have great practical advantages for anatomically complex structures such as the hypothalamus with regard to anatomical orientation, tissue storage and the study of archival material. In the last decade, our group has successfully developed radioactive ISH on formalin-fixed, paraffin-embedded human brain tissue sections to detect mRNA expression of e.g. vasopressin, oxytocin, tyrosine hydroxylase, corticotropin-releasing hormone (CRH), neuropeptide Y, agouti-related protein, and thyrotropin-releasing hormone [1], [2], [3], [4], [5], [6], [7], [8], [9]. However, when the standard ISH protocol was used for the detection of a gene of low-to-moderate abundance, background issues usually arise in the form of non-specific deposits of radioactivity, seen as speckles on autoradiographic film.

There are two sources of non-specific labeling of tissue following ISH: (1) cross-hybridization to related sequences other than the target, and (2) probe binding to non-RNA components in the tissue. While cross reaction can be limited or eliminated by a combination of careful probe design and manipulation of hybridization and washing conditions [10], interactions with non-RNA components are governed by chemical interactions which cannot be effectively addressed at times only by alteration of stringency. Therefore, optimizing ISH protocols represents a balance between conditions required for selective hybridization and conditions that limit probe binding to non-RNA components.

To address the background problems consisting of non-specific deposits, we introduced a number of alterations in the ‘routine’ protocol, illustrated here by the optimization procedure for quantitative ISH study of histidine decarboxylase (HDC) mRNA expression, the rate limiting enzyme for histamine production, in the human hypothalamic tuberomamillary nucleus (TMN), using paraffin-embedded brain tissue sections. The TMN in the posterior hypothalamus is the exclusive location of histaminergic neurons that send their fibers to almost all the regions of the human brain [11], [12], [13], [14]. As a neurotransmitter in the central nervous system, histamine holds a key position in the regulation of basic body functions, including attention, the sleep–wake cycle, energy and endocrine homeostasis, synaptic plasticity and learning [15]. In addition, the TMN may also play a role in brain diseases such as epilepsy, Alzheimer’s disease (AD), Parkinson’s disease (PD) [15]. Changes in the TMN are presumed to occur in PD on the basis of the extensive accumulation of the characteristic neuropathological PD lesions, i.e. the Lewy bodies and Lewy neuritis in this area [16], [17]. We aimed to optimize the protocol for reliably detecting changes in HDC-mRNA expression in the TMN of PD patients, using formalin-fixed, paraffin-embedded archival post-mortem human brain tissue. To this end, we introduced a few alterations into the routine protocol used in our group. We believe that this improved protocol has a general applicability for other low-to-moderate abundant genes in formalin-fixed, paraffin-embedded archival post-mortem human brain tissue, as shown here for the expression of CRH-mRNA in the hypothalamic paraventricular nucleus (PVN).

Section snippets

Materials and methods

A quantitative ISH study protocol can be divided into 5 consecutive stages: tissue preparation, probe labeling and purification, hybridization, post-hybridization washes, and autoradiographic detection. Variables involved in these different stages that might affect the results were tested alone or in combination. Based upon these tests, an improved ISH protocol was established and validated for film-quantification of HDC-mRNA expression in the TMN of PD patients and controls.

Specificity of HDC-mRNA in situ hybridization

Support for specificity of HDC-mRNA ISH signal came from the exclusive location of labeled cells in the TMN, observed on both film autoradiograms (Fig. 1A) and after emulsion autoradiography (Fig. 1B). Using emulsion autoradiography in combination with thionin-counterstaining, which enable signal analysis at the level of single cells, most of the characteristic, large TMN cells were masked with black silver grain deposits, showing medium to heavy labeling (Fig. 1B). In addition, specificity was

Discussion

The results of the present study demonstrate that the sensitivity of our previously developed ISH procedures using 35S-radioactively labeled oligonucleotides can be significantly improved by a few relatively simple alterations that facilitate detection of low-to-moderate gene expression, such as HDC-mRNA in the TMN, and other targets such as CRH-mRNA in the PVN of the human hypothalamus, using formalin-fixed, paraffin-embedded archival post-mortem tissues. The modifications not only resulted in

Acknowledgments

We thank the Netherlands Brain Bank for providing the brain material. We wish to thank Mr. B. Fisser for his technical assistance and Mrs. W.T.P. Verweij for correcting the English. Mr. Ling Shan was supported by the State Scholarship Fund (CSC) [2007]3020 from the China Scholarship Council. Dr. Ai-Min Bao was supported by the China Exchange Programme of the Royal Netherlands Academy of Arts and Sciences (KNAW) (project 09CDP011) and theHersenstichting Netherland (14F06(2).07).

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    These authors contributed equally to this paper.

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