Method Article

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

933 views

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 mM NaCl, 0.1% (v/v) Tween-20, pH 7.5.
  3. Dilute the two probes (DIG-labelled and FITC-labelled) 1/1,000 in hybridization buffer (DEPC-treated deionized water with 50% (v/v) deionized formamide, 200 mM NaCl, 5 mM EDTA, 10 mM Tris-HCl pH 7.5, 5 mM NaH2PO4, 5 mM Na2HPO4, 0.01 mg/ml yeast tRNA, 1× Denhardt's solution, and 10% w/v dextran sulfate) pre-warmed to 65 °C and mix well.
  4. Apply around 300 µl of hybridization mixture onto each slide, coverslip with oven-baked (200 °C) coverslips and incubate O/N at 65 °C in a humidified chamber.
  5. Transfer the slides into a Coplin jar containing pre-warmed wash buffer. Wash the slides for 30 min twice at 65 °C with wash buffer.
  6. Wash the slides for 10 min three times with MABT at RT.

2. Visualization of the FITC Probe

  1. Prepare ISH blocking buffer: MABT with 2% blocking reagent and 10% heat-inactivated sheep serum.
  2. Optional: use a hydrophobic pen to draw circles around the tissue sections on the slide to reduce the volume of antibody solutions required.
  3. Incubate the slides for 1 hr at RT with ISH blocking buffer in a humidified chamber.
  4. Incubate the slides O/N at 4 °C with a horseradish peroxidase (POD)- conjugated anti-FITC antibody diluted 1/500 (v/v) in ISH blocking buffer.
  5. Place the slides into a Coplin jar containing PBS with 0.1% (v/v) Tween-20 (PBST). Wash 3 times for 10 min in PBST and replace with fresh PBST each time.
  6. Immediately before use, prepare the fluorescent tyramide by diluting 100 times into the Amplification diluent. For this example, use FITC-tyramide.
  7. Add this to the slides and leave for 10 min at RT.
  8. Place the slides into a Coplin jar and wash 3 times in PBST for 10 min.

3. Visualization of the DIG Probe

  1. Prepare 0.1 M Tris- HCl pH 8.2.
  2. Incubate the slides with ISH blocking buffer for 1 hr at RT.
  3. Incubate the slides O/N at 4 °C with an alkaline phosphatase (AP) -conjugated anti-DIG antibody diluted 1/1,500 (v/v) in ISH blocking buffer.
  4. Wash with MABT for 10 min three times.
  5. Wash twice with 0.1 M Tris-HCl pH 8.2 for 5 min at RT.
  6. Immediately before use, prepare Fast Red solution by dissolving the tablets in 0.1 M Tris pH 8.2. Filter the solution through a 0.22 µM filter.
  7. Incubate the slides at 37 °C with Fast Red solution in a humidified chamber. As the time for optimal development varies, check the slides regularly with a fluorescent microscope to monitor the formation of precipitate. For this example, stop the reaction after 2 - 3 hr.
  8. Wash with PBST for 10 min three times.

Access restricted. Please log in or start a trial to view this content.

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

Reprints and Permissions

Request permission to reuse the text or figures of this JoVE article

Request Permission

Tags

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

Related Articles