ImmunohistochemistryRichard AbanesJune TseBICD 13008/15/2007Why we use antibody staining?-It is used to visualize the location of proteins within the embryo.-It can also be used to determine the function of a protein.Antigen vs Antibody► Antigen: a molecule that stimulates an immune response.► Antibody: a Y-shaped protein used by the immune system to identify and neutralize foreign objects.How to make primary antibodies?• Purify protein of interest and inject into host animal.• Give animal time to make antibodies for protein.• Extract/isolate antibodies from the animal.What are secondary antibodies?• They bind to the constant region of the primary antibody.• Fluorophores are usually attached to the constant region of the secondary antibodies. Protocol1. Fix specimen with 4% formaldehyde or methanol at room temperature.-fixatives are used to keep embryos at a certain stage of development2. Remove fixative and wash with PBS two times.3. Incubate in blocking buffer and then remove.-blocking buffer (contains fetal cow/bovine serum, Triton- X, and NaN3 ) is used to prevent non-specific bindingProtocol cont.4. Wash with PBS two times.5. Add primary antibodies and incubate at room temperature.Protocol cont.6. Remove primary antibodies.7. Rinse and wash with PBS two times.8. Add secondary antibodies.Protocol cont.9. Incubate in the dark at room temperature-incubating in the dark will prevent photobleaching10. Remove secondary antibodies and wash with PBS twice.11. Specimen is ready to be mounted on a slide and viewed by fluorescence microscope. How to assay the protein?-quantitative analysis of protein is determined by Western Blot
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