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Exercise caution when collecting, fixating and sectioning the samples. Glycerin gelatin should be used as the AEC mounting medium. For example, a labeled secondary antibody raised against rabbit IgG, which can be purchased "off the shelf," is useful with any primary antibody raised in rabbit. There are numerous IHC methods that can be used to localize antigens. Despite the shortcomings, the direct method is commonly applied to screen monoclonal antibodies before the large-scale manufacturing process.
Since DAB may cause skin and bladder cancers, it is advised that personal protective equipment should be used and skin/mucosa should be avoided. The secondary antibody must be raised against the IgG of the animal species in which the primary antibody has been raised. 34 40 With proper treatment, the section reveals clear tissue structure and exact antigen location to enable high medical-value pathology researches and retrospective studies. 0000002280 00000 n endstream endobj 35 0 obj<> endobj 37 0 obj<> endobj 38 0 obj<>/Font<>/ProcSet[/PDF/Text]/ExtGState<>>> endobj 39 0 obj<> endobj 40 0 obj<>stream As antigens may denature, disappear and diffuse, autopsy specimen should be fixated as soon as possible so as not to influence its label. Tissue samples are typically taken from specimens of various sources: biopsy, surgery, animal model and autopsy.
This is a process of treating the tissue in a paraffin box so that the paraffin wax cools down and solidifies. To avoid original protein structure restoring, do not cool the sample section by taking it out of the buffer solution. When either the horseradish peroxidase (HRP) or alkaline-phosphatase (AP) system is applied for IHC, activation of endogenous enzymes should be blocked or inhibited to avoid producing non-specific binding. Avidin-Biotin Peroxidase Complex (ABC) and Labeled Streptavidin Binding (LSB) are the two most widely used affinity methods for amplifying the target antigen signal. Hematoxylin, methyl green and methyl blue are the compatible counterstains. Please refer to the Fixation section described above. It helps keep the cross-linking between tissues and maintain antigen. Carbodiimide, dimethylacetamide, dimethyl-suberimidate, para-benzoquinone are widely used in tissue fixation of peptide hormones. There are four levels for fluorescence intensity: Accurate antigen location is enabled with better contrast ratio, Stained samples can be stored for a long time, Hematoxylin can be used as counterstain which enhances study of tissue morphology, End-product color can be easily identified and observed by light microscope (and also by electron microscopy due to high electron density), Double and multiple stains can be implemented, Specific antiserum for the tissue antigen (AnTAn), Antiserum against the immune globulin of the species for AnTAn, Specific antiserum prepared against the enzyme label in the same species as AnTAn, Incubation of primary antibody with tissue sample to allow binding to target antigen, Incubation of biotinylated secondary antibody (which has specificity against primary antibody) with tissue sample to allow binding to primary antibody, Pre-incubation of biotinylated enzyme (HRP or AP) with free avidin to form large ABC complexes (Biotinylated enzyme and avidin are mixed together in a pre-determined ratio to prevent avidin saturation), Incubation of the above pre-incubated solution to tissue sample, Incubation of streptavidin-enzyme conjugate to tissue sample.