Immunohistochemistry
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| antigen/immunogen | any substances that can induce a detectable immune response; proteins best antigens (polysaccharides, nucleic acids and other polymers can act antigenetic); most common: BACTERIA, VIRUSES
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| antibody/immunoglobulins (Ig) | proteins produced by B lymphocytes in response to antigenic stimulation
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| substrate | the substance on which an enzyme acts (peroxide for peroxidase, and naphthol-AS-phosphate for phosphatase)
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| chromogen | a BENZENE dirivative containing a color-bearing group(chromophore)
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| fluorochrome | dye that absorbs light and then emits its own light at a longer wavelength(fluorescence) used in IF (kidney & skins)
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| epitope | antibodies bind to these short regions of the specific antigen; each antibody binds to a different epitope
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| secondary antibody | an antibody that binds to primary antibodies or antibody fragments. They are typically labeled with probes that make them useful for detection, purification or cell sorting applications
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| multilink antibody | cocktails of antibodies raised in different species; mixtures of biotinylated anti-mouse, anti-rabbit and many more; similar to universal link antibody binds to a primary antibody made in any of a variety of animal species or to both poly and momoclonal
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| Polyclonal antibody | mixture of a "pool" or antibodies from many clones of lymphocytes; difficult to standardize; limited uses; can be pooled from many immunized animals of the same species = less likely to exhibit major batch-to-batch variations
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| Monoclonal antibody | from cloned hybridoma cells capable of producing antibody that is identical to the original; can be characterized, standardized and produced in unlimited quantities; homogeneity, absence of nonspecific Igs, purer, no diff in batches; lack of background
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| Direct | labeled (fluorscent dye or enzyme for subsequen reaction w/ a chromogen) antibody of known specificity to used to identify antigens
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| Indirect | patient's serum added to tissue sections containing known antigens to test for presence of antibodies; method using two antibodies to detect antigen in patient's tissue
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| Two-step indirect | 1st antibody, unlabeled defined antibody & 2nd antibod, LABELED used to localize 1st antibody; used w/ enzyme-conjugated antibodies
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| Three-step indirect | both 2nd and 3rd antibodies are labeled with enzymes, good means of increasing staining intensitive; used w/ enzyme-conjugated antibodies
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| Peroxidase-Antiperoxidase | PAP complex is composed of enzyme peroxidase and antibody against peroxidase; peroxidase in presence of HOOH and chromogen forms a colored compound
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| Alkaline phosphatase-antialkaline phosphatase | enzyme IHC demonstrated by naphthol-AS-phosphate and chromogen, alkaline phosphatase combines the two to make colored compound
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| Avidin-Biotin Complex | ABC, primary antib is followed by biotinylated 2ndary antibody (linking) 3rd step is application fo apreformed Avidin-biotin enzymes; low background, economy and sensitived; antibodies may be used in high dilution
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| Labeled Avidin-Biotin complex | LAB; primary antib is followed by biotinylated 2ndary antibody (linking) 3rd step is application fo apreformed Avidin-biotin enzymes; 3rd, application of enzymes labeled avidin; 4-8x more sensitive than ABC
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| List fluorochromes | fluorescein isothyoicyanate (FITC) & rhodamine
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| Two common chromogens used w/ immunoperoxidase techniques | DAB and 3-animo-9ethycarbazole (AEC-soluble in organic solvents and require aqueous mounting)
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| Identify two chromogens used with alkaline phosphatase techniques | fast read violet LB, fast red Tr, or fast blue BBN; soluble in organic solvents and require aqueous mounting medium
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| Identify three enzyme systems used in enzyme immunohistochemistry | horseradish peroxidase, alkaline phosphatase, glucose oxidase (also beta galactosidase)
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| Preferred method of specimen preparation for immunofluorescence | frozen sections of unfixed tissue, soluble antigens are lost; fixation and processing impair antigenic reactivity (can dehydrate or use acetone-formalin, but still; nonadditive, coagulant fixatives good-acetone, ROH); zinc formalin GOOD immunoreactivity
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| Problems that may be encoutered with formaldehyde fixation of specimens for immunoperoxidase | as a noncoagulating additive, cross-links proteins and impairs antigenic reactivity by hiding the reactive sites
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| Fixatives other than formalin and describe special uses | aceton/roh (coag,nonadd): good penetration of antibody, do not block immunoreactive determinants; HgCl (coag, add) (B-5) good for intracytoplasmic antig, surface membranes not good, good for LN; Zenker/Bouin/Roh-based good; glutaraldehyde = NO
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| Two blocking reactions and identify purpose of each | hydrogen peroxide (100% MetOH)--blocks tissue endogenous peroxidase activity (lots of RBC); addition of innocuous protein solution to tissue before 1st antibody so that it binds to charged (ie collagen, conn.tiss) sites eliminating nonspecific binding
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| Five heavy chains and two light chains that make up the five major classes of immunoglobulins | 2 identical heavy: gamma, alpha, mu, delta, episilon; 2 identical lige: kappa or lambda; IgG, IgA, IgM, IgD, IgE
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| Preferred method of specimen preparation if a panel of lymphocyte surface-marking antibodies is needed | use Zinc Formalin, not B-5 unless you only want to stain cytoplasmic Igs
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| state method of preparation of negative controls | use tissue that's expected to be negative for the antibody (no antigen), stain using same kit (specific); use patient tissue processed the same way as patient sample, and process/stain w/ DILUENT w/o antibody; DETECTS UNINTENDED BACKGROUND STAINS
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| Six problems that may occur with immunohistochemical staining and state a corrective action for each | Specimen unstained, positive unstained; specimen unstained, positive stained; specimen weak, positive weak; specimen weak; positive stained; specimen excessive background, pos excessive background; specimen excessive background, control no background
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| Reason that routine positive controls should be prepared in your laboratory and not purchased | because fixation varies so much and affects rxns and overfixation can occur (store bought wouldn't show this); positive controls must be treated w/ exact same steps so as to show no false-positive staining, good comparison to the standardized
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| Two methods of epitope enhancement (antigen retrieval) | HIER - heat induced epitope retrieval; EIER - enzyme induced epitope retrieval
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| List solutions that have been used for heat-induced epitope enhancement | immersing formalin fixed tissue in metallic salt solutions:saturated led thyocyanate/1% zinc sulfate (toxic), Na-citrate buffer (.01M/6.0), distilh2o/L glycine HCL(3.5), 3M urea soltuion; high pH good 8-9; but can break cross-links
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| three ways of heating in the heat-induced epitope enhancement methods (HIER) | microwave/pressure cooker combines greastest effectiveness; autoclave gives good results; pressure cooker heated w/ hot plate
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| three solutions that have been used for enzyme-induced epitope enhancement (EIER) | older; TRYPSIN in 0.1% CaCL2; 0.1% trypsin in PBS; other enzymes used pronase in TRIS buffer, pH7.5; ficin; 0.1% progease in PBS, 7.4; 0.4% pepsin in 0.1N HCL; can be dependent on location of epitope
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| Which chromogens are alcohol soluble and which are not | all of the chromogens (AEC, rast red-violet LB, fast red TR or fast blue BBN) except DAB are soluble in organic solvents and require aqueous mounting media; hence hematoxylin solutions w/ ROH cannot be used, false-negatives will result
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| Four chemicals used to intensify the 3,3'-diaminobenzidine (DAB) reaction | heavy medals (NiCl, CuCl, CoCl) rinse of increasing background stain; Imidazole (0.1M,7.6pH) superior, inhibits Hg activity; OsO4 FOLLOWING DAB rxn, prevents fading but can darken background staining as well as intensify
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| Unlabeled or soluble enzyme immune complex | 3-sted method using primary antibody, linking/secondary antibody, and soluble enzyme-antienzyme complexes; 1st and 3rd need to made in same animals species for 2nd to link; PAP or APAP
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| Avidin | high affinity for Biotin and the binding is essentially irreversible
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| advantages of epitope enhancement/antigen retrieval | ability to further dilute antibodies; more intense rxns, decrease incubation; more uniform stain; decreased bkgrnd stain; daily consistency; better standardization
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| disadvantages to HEIR | tissue sections can be damaged in heating in strongly basic pH; can cause loss of tissue, burns and destruction of antigenicity; some reagents toxic, heat under a fume hood
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| disadvantages to EIER | enzyme digestion usually reduces nonspecific, but can AL INCREASE nonspecific staining, can also weaken specific staining = false-negatives; cause fragmentation or loss of tissue sections; only a few antibodies work well with
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| use of multilink antibodies |
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Created by:
Miellee
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