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 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.

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