FDA免疫组织化学治疗目标识别：整体检测系统.pptVIP

Immunohistochemistry for Therapeutic Target Identification: The Total Test System Timothy J. O’Leary Armed Forces Institute of Pathology Proteases Destroy protein antigens Not all protein antigens are destroyed at the same rate Nucleases Destroy nucleic acids RNA destroyed much more quickly than DNA Found in different levels in different tissues. For example, pancreas and eosinophils have extremely high levels of ribonucleases Stability In general DNAProteinRNA Some proteins are essentially as stable as DNA, however. Fixation Stops degradation of cellular components by enzymes Preserves cellular morphology Ideal Fixative An inexpensive substance that rapidly penetrated any tissue and inactivated destructive enzymes, preserving cell and tissue structure, without reducing antigenicity, the ability to extract cellular components for other analyses, or the ability to use cellular nucleic acids as substrates for polymerase reactions…. Fixatives Cross-linking, such as formaldehyde Precipitating, such as alcohol Cross-Linking Fixatives Intramolecular and intermolecular cross-links in proteins Cross links between DNA bases, but only in non-double stranded areas Adducts between RNA and formaldehyde (methylol), proteins, nucleic acids Intermolecular bonds in some lipids Fixatives and the Pathologist Most pathologists prefer formaldehyde for most tissues - relatively “crisp” tissue morphology, with relatively good preservation of antigenicity. Other fixatives include alcohols, acetone, Zenkers, Bouins, B5, zinc-formalin, etc…. Formaldehyde Fixation and Protein Antigens Fixation may reduce antigen reactivity in immunohistochemical reactions by masking epitopes or by making the tissue inpenetrable by antibodies. Fixation does not destroy secondary structure. Fixation partially reversible, but…. Antigen Retrieval Generally some variant on heating a slide in solution to a high temperature and incubating it. Removes formaldehyde, which forms a relatively weak bond with