Method for Whole Mount Antibody Staining in Chick

Summary

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

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 antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell ¹.

Protocol

I. Schematic Overview:

This video demonstrates the different steps in whole mount immunohistochemistry in chick embryo. First, the embryo is fixed in PFA [IHC1]. Then, endogenous peroxidase activity is quenched [IHC2]. The embryo is then incubated in primary antibody [IHC3]. After several washes, the embryo is incubated in secondary antibody [IHC4]; Color reaction is revealed using DAB [IHC5] and antibody staining appears orange [IHC6].

Discussion

This video demonstrates the different steps in performing whole-mount antibody staining in young chick embryos. This protocol is essentially used for the spatial and temporal characterization of novel antibodies in chick ^2,3, as well as for the use of known antigenic markers to determine embryonic malformations following insult ⁴.

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Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Eggs		Charles River Laboratories	Premium Fertile	Fertilized, HH4 (16 hr)
Stereomicroscope	Microscope	Leica Microsystems	MZ9.5 or similar
Marsh Automatic Incubator	Other	Lyon	RX
Curved Forceps (1)	Tool	Electron Microscopy Sciences	72991-4C
Forceps (2)	Tool	Fine Science Tools	11002-13
Fine scissors	Tool	Fine Science Tools	14161-10
Plastic dishes	Tool	Falcon BD	353001
Rubber Bulb	Tool	Electron Microscopy Sciences	70980
Pasteur Capillary Pipette	Tool	Electron Microscopy Sciences	70950-12	round edge under flame
Microdissecting knife	Tool	Fine Science Tools	10056-12	Use to cut embryo from surrounding membranes following fixation
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
16% PFA	Reagent	Electron Microscopy Sciences	15710
30% H2O2	Reagent	Sigma-Aldrich	H1009
BSA	Reagent	Sigma-Aldrich	A3803
NGS	Reagent	Jackson ImmunoResearch	005-000-121
Primary Antibodies	Reagent	Developmental Studies Hybridoma Bank	4G11, 15.3B9, PAX7	For these antibodies, investgators used mouse donnors
Peroxidase conjugated-Goat Anti-Mouse IgG (H+L)	Reagent	Jackson ImmunoResearch	115-035-003
3,3’-diaminobenzidine tetrahydrochloride	Reagent	Pierce, Thermo Scientific	34001	Store at -20; Allow bottle to warm to RT before use.
Cedar wood oil	Reagent	Sigma-Aldrich	W522503
Fast green FCF	Reagent	Sigma-Aldrich	F7252
Minuten pins 0.2mm diam		Fine Science Tools	26002-20