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

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....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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	09:20
α-FITC POD-conjugated antibody	Roche	11426346910
TSA™ Plus Fluorescein System	Perkin Elmer	NEL741001KT	26:00:00
α-DIG AP-conjugated	Roche	11093274910
Fast red tablets	Roche	11496549001
Mounting medium	Dako	S3023
Leica SP2 confocal microscope	Leica
.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 dihydrate	Sigma	30435
Yeast tRNA	Roche	10109495001
50x Denhardt's solution	Life Technologies	750018
Dextran sulfate	Sigma	D8906
Sodium acetate	Sigma	S2889
Trizma Base	Sigma	T1503