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Neuroscience

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Published: March 26th, 2016

DOI:

10.3791/53976

1Wolfson Institute for Biomedical Research, University College London
* These authors contributed equally

Localizing gene expression to specific cell types can be challenging due to the lack of specific antibodies. Here we describe a protocol for simultaneous triple detection of gene expression by combining double fluorescence RNA in situ hybridization with immunostaining.

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can be hampered by the lack of specific primary or secondary antibodies for immunostaining. Here we describe a protocol to detect the expression of three different genes in the same brain section using double fluorescence in situ hybridization with two gene-specific probes followed by immunostaining with an antibody of high specificity directed against the protein encoded by a third gene. The Aspartoacyclase (ASPA) gene, mutations of which can lead to a rare human white matter disease - Canavan disease - is thought to be expressed in oligodendrocytes and microglia but not in astrocytes and neurons. However, the precise expression pattern of ASPA in the brain has yet to be established. This protocol has allowed us to determine that ASPA is expressed in a subset of mature oligodendrocytes and it can be generally applied to a wide range of gene expression pattern studies.

Glial cells, which are the most abundant cells in the central nervous system (CNS), comprise oligodendrocytes (the myelinating cells of CNS), oligodendrocytes precursors (OPs, also known as "NG2 cells"), astrocytes and microglia. There is growing interest in the functions of glial cells and their potential roles in neurological diseases1. For example, Canavan disease (CD) is a hereditary neurodegenerative disorder starting early in infancy with spongiform leukodystrophy and a progressive loss of neurons, leading to death usually before 10 years of age2,3. Mutations in the Aspartoacyclase (ASPA) gene that lead to drastica....

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Ethics Statement:
Mouse husbandry and handling are in accordance with UK Home Office regulations and UCL ethics committee guidelines, complying with the Animals (Scientific Procedures) Act 1986 of the United Kingdom and its Amendment Regulations 2012.

NOTE: All solutions should be made with diethyl pyrocarbonate (DEPC)- treated water to destroy any residual RNase. For DEPC treatment, add DEPC (1 ml per litre), shake vigorously until all the DEPC globules have disappeared then autoclave t.......

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This article describes a method for a double fluorescence ISH followed by immunolabelling in mouse brain sections. A brief description of this protocol is shown in Figure 1. The first step was to synthesize probes specific to Aspa and Mbp (myelin basic protein). To check that the probes had been synthesized, a small aliquot of each reaction was run on an agarose gel. The faint linear template and a large amount of the RNA probe can be seen (.......

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This protocol provides a step-by-step procedure for a double RNA in situ hybridization followed by immunostaining. We have used this protocol to confirm that Aspa is expressed in mature oligodendrocytes in several brain areas.

This multi-step procedure has many potential pitfalls that can affect sensitivity and should be avoided. First, all the solutions and storage buffers for the transcription reaction need to be RNase-free. Second, the choice of cDNA templates is important.......

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Work in the authors' laboratories was supported by the UK Biotechnology and Biological Sciences Research Council (BB/J006602/1 and BB/L003236/1), the Wellcome Trust (WT100269MA) and the European Research Council (ERC, "Ideas" Programme 293544). SJ was supported by an EMBO long-term fellowship. The authors thank Stephen Grant for his technical assistance.

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Name Company Catalog Number Comments
QIAprep® Miniprep Qiagen 27104
Deionized formamide Sigma F9037 for ISH blocking buffer
Sodium chloride Sigma S3014
Trizma Base Sigma T1503
Hydrochloric acid VWR International 20252.290
Sodium phosphate monobasic anhydrous Sigma S8282
Sodium phosphate dibasic dihydrate Sigma 30435
Yeast tRNA Roche 10109495001
50x Denhardt's solution Life Technologies 750018
Dextran sulfate Sigma D8906
Aspa cDNA clone Source Bioscience IRAVp968C0654D
SalI New England Biolabs R0138
Sodium acetate Sigma S2889
Equilibrated phenol Sigma P4557
Chloroform Sigma-Aldrich C2432
Isoamyl alcohol Aldrich 496200
Ethanol VWR International 20821.321
T7 RNA polymerase Promega P4074
Transcription buffer Promega P118B
100mM DTT Promega P117B
UTP-DIG NTP mix Roche 11277073910
Rnasin Promega N251B
Paraformaldehyde Sigma P6148
Filter paper Fisher scientific 005479470
Sucrose Sigma 59378
Diethyl pyrocarbonate Sigma D5758
Pentobarbitone Animalcare Ltd BN43054
Dissecting scissors World Precision Instruments 15922
25 gauge needle Terumo 300600
Peristaltic pump Cole-Parmer Instrument Co. Ltd WZ-07522-30
Iris scissors Weiss 103227
No.2 tweezers World Precision Instruments 500230
Coronal Brain Matrix World Precision Instruments RBMS-200C
Razor blade Personna Medical PERS60-0138
OCT medium Tissue tek 4583
Cryostat/microtome Bright
Superfrost plus slides Thermo Scientific J1800AMNZ
Sodium citrate Sigma S4641 for 65°C wash buffer
Formamide Sigma-Aldrich F7503
Tween-20 Sigma-Aldrich P1379
Coverslips VWR International 631-0146
Coplin Jar Smith Scientific Ltd 2959
Blocking reagent Roche 11096176001
Heat-inactivated sheep serum Sigma S2263
Hydrophobic pen Cosmo Bio DAI-PAP-S 1:500
α-FITC POD-conjugated antibody Roche 11426346910
TSA™ Plus Fluorescein System Perkin Elmer NEL741001KT 1:1500
α-DIG AP-conjugated Roche 11093274910
Fast red tablets Roche 11496549001
.22µM filter Millex SLGP033RS
α-Olig2 Rabbit antbody Millipore AB9610
Alexa Fluor® 647-conjugated α-rabbit antibody Life technologies A-31573 1:1000
bisBenzimide H 33258 sigma B2883
Mounting medium Dako S3023
Leica SP2 confocal microscope Leica

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