Double Whole Mount in situ Hybridization of Early Chick Embryos

Summary

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

Abstract

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC. Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

Protocol

Part 1: Fixing the embryos

1. Fill dissection dish with ice cold depc-PBS. Keep on ice. Open the egg by tapping the shell with forceps and remove shell pieces.
2. Remove thick albumin with forceps. Tilt yolk sac with coarse forceps so that embryo faces upwards.
3. Using scissors, cut a square of yolk sac around the embryo.
4. Using spoon, remove embryo from yolk and place on ice cold depc-PBS.
5. Under the microscope, remove membranes and yolk and transfer to fixation dish.
6. Pin down, remove depc-PBS. Replace with 4% PFA in depc-PBS and fix O/N at 4°C.
7. Remove fixative. Replace with ....

Discussion

The 2-color whole mount in situ hybridization method is used to determine both spatial and temporal patterns of gene expression, using one or two genes of interest. Applications include assessment of gene expression patterns of novel genes (e.g. ^1,2, as well as changes in gene expression following insult (embryonic manipulations ³, beads ⁴, and electroporation of RNA or DNA constructs ⁵).

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Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Eggs	Animal	Charles River Laboratories	Premium Fertile
Stereomicroscope	Microscope	Leica Microsystems	MZ9.5
Marsh Automatic Incubator	Tool	Lyon	RX
Hybridization Incubator	Tool	Hybaid	Micro-4
Adams Nutator orbital mixer	Tool	Fisher Scientific	14-062
Curved Forceps (1)	Tool	Electron Microscopy Sciences	72991-4C
Fine scissors	Tool	Fine Science Tools	14161-10
Spoon	Tool	Fine Science Tools	10370-18
Pasteur Capillary Pipette		Electron Microscopy Sciences	70950-12
Microcapillary tube		Sigma-Aldrich	P1049-1PAK	Pull using vertical micropipette puller; blunt end with fine forceps
Microdissecting knife		Fine Science Tools	10056-12	Use to puncture cavities prior to in situ hybridization
Minuten pins 0.2mm diam		Fine Science Tools	26002-20	Keep pins together using a magnetic stirrer;
Sylgard 184 Silicon Elastomer Curing Agent and Base	Reagent	Dow Corning	0001986475	Mix 1 part Curing Agent, 9 parts Base; set O/N at 37C
Diethylpyrocarbonate (depc)	Reagent	Acros Organics	10025025	Add 1ml depc to 1l PBS; shake; autoclave
16% PFA	Reagent	Electron Microscopy Sciences	15710
Tween-20	Reagent	Sigma-Aldrich	P1379-100ML
Proteinase K		Sigma-Aldrich	P2308-5MG	Stock at 10 mg/ml in depc-H2O in 10ul aliquots. Store at -20C.
Formamide, deionized	Reagent	Ambion	9342
Yeast tRNA-25 mg	Reagent	Invitrogen	15401-011	Stock at 20 mg/ml in depc-H2O in 125ul aliquots. Store at -20C
Heparin Sodium salt	Reagent	Sigma-Aldrich	H4784-250MG	Stock at 50 mg/ml in depc-H2O in 100ul aliquots. Store at -20C.
SSC x20 pH5.5	Reagent	Fisher Scientific	PR-V4261
EDTA (0.5M)	PR-V4231	Fisher Scientific
SDS	Reagent	Fisher Scientific	BP166-100
Maleic acid	Reagent	Fluka	63189
B–hringer Blocking Reagent	Reagent	Roche Group	11096176001	Use at 2% in MABT. To make, heat with MABT for 20 mn at 60C. Can make a stock and store in 1-5 ml aliquots at 20C.
Heat inactivated sheep serum	Reagent	Jackson ImmunoResearch	013-000-121	Dissolve in 10 ml H2O; heat 550C, 30 mn. Aliquot at 1ml, -20C.
Anti-Digoxigenin-AP, Fab fragments	Reagent	Roche Group	11093274910
NBT/BCIP stock solution	Reagent	Roche Group	11681451001
Anti-Fluorescein-AP, Fab fragments	Reagent	Roche Group	11426338910
2M Tris	BP1759-500	Fisher Scientific
Vector Red Alkaline Phosphatase Substrate Kit	Reagent	Vector Laboratories	SK-5100
1,2,3,4-tetrahydronaphthalene	Reagent	Sigma-Aldrich	429325-100ML	Under hood