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Immunogold Electron Microscopy01:20

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Immunocytochemistry (ICC) and immunohistochemistry (IHC) are techniques that use antibodies to check for specific proteins or antigens in a sample. The technique was first published by Albert Coons in 1941 to detect the presence of pneumococcal antigen in tissue sections from mice infected with Pneumococcus. Immunocytochemistry helps localization of proteins or antigens in individual cells like blood cells, stem cells, etc., while immunohistochemistry does the same for tissue samples.
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An artificial test substrate for evaluating electron microscopic immunocytochemical labeling reactions.

G D Gagne, M F Miller

    The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society
    |August 1, 1987
    PubMed
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    A novel artificial substrate system optimizes electron microscope immunocytochemistry. This method allows separate evaluation of fixation and embedding effects on antigen labeling, improving accuracy in studies of hepatitis B surface antigen (HBsAg).

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    Area of Science:

    • Biochemistry
    • Microscopy
    • Immunology

    Background:

    • Electron microscope immunocytochemistry requires precise optimization of labeling parameters.
    • Conventional methods can confound the effects of fixation and embedding on antigen detectability.

    Purpose of the Study:

    • To develop and validate an artificial substrate system for optimizing electron microscope immunocytochemical labeling parameters.
    • To quantitatively determine optimal fixation and embedding conditions for specific antigens.

    Main Methods:

    • Incorporation of antigens into glutaraldehyde-polymerized BSA blocks.
    • Fixation, embedding, and immunogold labeling of the artificial substrate, mimicking tissue processing.
    • Evaluation of labeling parameters including antibody concentration and fixation/embedding procedures.

    Main Results:

    • The BSA substrate system successfully allowed separate evaluation of fixation and embedding effects.
    • Optimal conditions were determined for labeling hepatitis B surface antigen (HBsAg), human IgG, and horseradish peroxidase.
    • Unfixed and unembedded HBsAg was detectable at concentrations below 20 µg/ml.
    • Conventional aldehyde fixation protocols prevented HBsAg labeling in resin-embedded cells.

    Conclusions:

    • The artificial substrate system provides a robust method for optimizing immunocytochemical labeling parameters.
    • Conventional fixation and embedding procedures are unsuitable for labeling certain antigens, such as HBsAg, within resin-embedded cells.