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HT/HTL:Chap12

Immunohistochemistry

QuestionAnswer
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
antibody/immunoglobulins (Ig) proteins produced by B lymphocytes in response to antigenic stimulation
substrate the substance on which an enzyme acts (peroxide for peroxidase, and naphthol-AS-phosphate for phosphatase)
chromogen a BENZENE dirivative containing a color-bearing group(chromophore)
fluorochrome dye that absorbs light and then emits its own light at a longer wavelength(fluorescence) used in IF (kidney & skins)
epitope antibodies bind to these short regions of the specific antigen; each antibody binds to a different epitope
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
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
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
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
Direct labeled (fluorscent dye or enzyme for subsequen reaction w/ a chromogen) antibody of known specificity to used to identify antigens
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
Two-step indirect 1st antibody, unlabeled defined antibody & 2nd antibod, LABELED used to localize 1st antibody; used w/ enzyme-conjugated antibodies
Three-step indirect both 2nd and 3rd antibodies are labeled with enzymes, good means of increasing staining intensitive; used w/ enzyme-conjugated antibodies
Peroxidase-Antiperoxidase PAP complex is composed of enzyme peroxidase and antibody against peroxidase; peroxidase in presence of HOOH and chromogen forms a colored compound
Alkaline phosphatase-antialkaline phosphatase enzyme IHC demonstrated by naphthol-AS-phosphate and chromogen, alkaline phosphatase combines the two to make colored compound
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
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
List fluorochromes fluorescein isothyoicyanate (FITC) & rhodamine
Two common chromogens used w/ immunoperoxidase techniques DAB and 3-animo-9ethycarbazole (AEC-soluble in organic solvents and require aqueous mounting)
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
Identify three enzyme systems used in enzyme immunohistochemistry horseradish peroxidase, alkaline phosphatase, glucose oxidase (also beta galactosidase)
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
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
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
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
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
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
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
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
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
Two methods of epitope enhancement (antigen retrieval) HIER - heat induced epitope retrieval; EIER - enzyme induced epitope retrieval
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
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
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
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
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
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
Avidin high affinity for Biotin and the binding is essentially irreversible
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
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
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
use of multilink antibodies
Created by: Miellee