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].
Part 1: Fixing the embryos
Part 2: Preparing embryos for antibody step
Part 3: Antibody incubation
Part 3: Color reaction
Part 4: Embryo processing for photography and histology
Representative Results:
In the examples shown below, embryos are dissected at stages HH 10 (A), 12 (B) and 11 (C); Embryos show expression of PAX 7 in emerging neural crestas well as in somites and neural tube (A,B); In (C), notochord is labelled with 15.3B9 (Not-1) (Antibodies provided by Developmental Studies Hybridoma Bank).
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Eggs | Charles River Laboratories | Premium Fertile | Fertilized, HH4 (16 hr) | |
Stereomicroscope | Microscope | Leica Microsystems | MZ9.5 or similar | |
Marsh Automatic Incubator | 기타 | 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 | 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 | H1009 | |
BSA | Reagent | Sigma | A3803 | |
NGS | Reagent | Jackson Immuno | 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 Immuno | 115-035-003 | |
3,3’-diaminobenzidine tetrahydrochloride | Reagent | Pierce | 34001 | Store at -20; Allow bottle to warm to RT before use. |
Cedar wood oil | Reagent | Sigma | W522503 | |
Fast green FCF | Reagent | Sigma | F7252 | |
Minuten pins 0.2mm diam | Fine Science Tools | 26002-20 |