Method Article

Performing Double Fluorescence In Situ Hybridization to Detect Gene Expression in Mouse Brain Sections

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April 28th, 2025

In This Article

Abstract

Source: Jolly, S., et.al. Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections. J. Vis. Exp. (2016).

This video demonstrates the use of double fluorescence in situ hybridization to visualize the expression of two RNA targets in mouse brain sections. The protocol involves sequential detection of RNA using probes labeled with fluorescein isothiocyanate and digoxigenin, followed by enzymatic signal amplification to enhance sensitivity. The resulting fluorescence enables co-localization analysis of the target RNAs within the tissue.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

1. Hybridization

  1. Prepare 65 °C wash buffer: 150 mM NaCl, 15 mM Na3C3H5O(COO)3 (= 1x saline sodium citrate), 50% (v/v) formamide and 0.1% (v/v) Tween-20.
  2. Prepare MABT buffer: 100 mM maleic acid, 150 m....

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Superfrost plus slidesThermo ScientificJ1800AMNZ
Sodium citrateSigmaS4641for 65°C wash buffer
FormamideSigma-AldrichF7503
Tween-20Sigma-AldrichP1379
CoverslipsVWR International631-0146
Coplin JarSmith Scientific Ltd2959
Blocking reagentRoche11096176001
Heat-inactivated sheep serumSigmaS2263
Hydrophobic penCosmo BioDAI-PAP-S09:20
α-FITC POD-conjugated antibodyRoche11426346910
TSA™ Plus Fluorescein SystemPerkin ElmerNEL741001KT26:00:00
α-DIG AP-conjugatedRoche11093274910
Fast red tabletsRoche11496549001
Mounting mediumDakoS3023
Leica SP2 confocal microscopeLeica
.22 μM filter Millex SLGP033RS
Deionized formamide Sigma F9037 for ISH blocking buffer
Sodium chloride Sigma S3014
Hydrochloric acid VWR International 20252.29
Sodium phosphate monobasic
anhydrous
Sigma S8282
Sodium phosphate dibasic dihydrateSigma 30435
Yeast tRNA Roche 10109495001
50x Denhardt's solution Life Technologies 750018
Dextran sulfateSigma D8906
Sodium acetate Sigma S2889
Trizma Base Sigma T1503

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Tags

Double FISHRNA Probe DetectionSignal AmplificationCo localization AnalysisFITC TyramideFast Red SolutionAlkaline PhosphataseHorseradish Peroxidase

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