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The following general protocol is intended for use as a guideline in developing antibody-specific procedures. Different antibodies and tissues may require changes to this procedure. Review of individual product datasheets and relevant literature references may be helpful in customizing this procedure for specific applications.
Single-protein detection (specificity) on western blots is an absolute requirement. It is strongly advised that the researcher test each antibody on a western blot of the tissue they intend to use, to control for differences between tissues. Once the western blot is verified, the antibody can be tested on tissue sections, with negative controls consisting of no primary antibody (to see if there is direct staining by the secondary antibody), and a no secondary antibody control (to see if the primary antibody contributes endogenous peroxidase activity, or autofluorescence).
Since the protocols below are general, it is highly recommended to review the methodology and variations in IHC protocols; Immunocytochemical Methods and Protocols (second edition), edited by Lorette C. Javois, from Methods in Molecular Medicine, volume 115, Humana Press, 1999 (ISBN 0-89603-570-0).
Immunocytochemistry (ICC), by definition is the demonstration of a tissue constituent in situ by detecting specific antibody-antigen interactions where the antibody has been tagged with a visible label. The visual marker may be a fluorescent dye, colloidal metal, hapten, radioactive marker or the more commonly light microscopy an enzyme. Experimental samples ranging from frozen sections, cell culture/suspension, to whole tissue samples have been used. Ideally, maximal signal strength along with minimal background or non-specific staining are required to give optimal antigen demonstration. It is recommended to review the methodology and variations in protocols from; Immunocytochemical Methods and Protocols (second edition), edited by Lorette C. Javois, from Methods in Molecular Medicine, Humana Press, 1999 (ISBN 0-89603-570-0).
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