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IHC
Staining
| Question | Answer |
|---|---|
| Antigen | immunogen, induces immune response, proteins best, may be polysaccharides, nucleic acids, polymers, bacteria & viruses most common |
| Antibody | immunoglobulin, proteins prod'd by β lymphocytes, buy as pre-diluted working soln. (less stable) or conc'd stock, have 2 short light chains (Κ, λ) - react w/ antigen, & 2 heavy chains (γ , Δ, α, μ , ε) |
| 5 classes of antibodies | Immunoglobulin G (IgG) γ Immunoglobulin A (IgA) α Immunoglobulin M (IgM) μ Immunoglobulin D (IgD) Δ Immunoglobulin E (IgE) ε |
| Epitope | Region of antigen antibodies bind, many antibodies can bind same antigen, each antibody binds one epitope |
| Polyclonal antibody | Pool of antibodies from many clones of lymphocytes |
| Monoclonal antibodies | Primarily made in mice, so link usu. anti-mouse, high homogeneity, nonspecific antibodies absent, little batch-to-batch variability |
| 4 methods of IHC staining | Direct, indirect, unlabeled or soluble enzyme immune complex, avidin-biotin |
| Direct method | Known, labeled antibody used to ID tissue antigens |
| What can be used to label an antibody? | Fluorescein isothiocyanate (FITC), fluorescent dye, enzymes (horseradish peroxidase, alkaline phosphatase, glucose oxidase) - react w/ chromagen |
| Indirect method | Serum added to soln. w/ known antigens, labeled antibody used to detect bound antibodies from serum |
| 2-step indirect method | Technically direct, 2° labeled antibody used to localize 1° unlabeled defined antibody |
| 3-step indirect method | 2nd & 3rd antibodies labeled w/ enzymes, good to incr. staining intensity |
| 2- & 3-step indirect methods are usually used w/: | Enzyme-conjugated antibodies for light microscopy, multi-step indirect enhances tissue staining for light microscopy |
| Unlabeled or soluble enzyme immune complex method | 3-step method, 1° & soluble enzyme-antienzyme complexes (same species), 2° linking antibody, usu. PAP or alkaline phosphatase-antialkaline phosphatase |
| Avidin-biotin methods are: | Avidin-biotin complex (ABC) & labeled avidin-biotin (LAB) methods, 1° followed by biotinylated linking 2°, then avidin biotin-enzyme complex (ABC), or enzyme-labeled avidin (LAB) |
| Avidin | High affinity for biotin, originally from eggwhites, now use streptavidin from Streptomyces avidinii |
| Immunofluorescence staining | Most common FITC & rhodamine, when fluorochrome attached to antibody rxn sites easily visualized |
| Best hematoxylin counterstain for IHC? | Mayer b/c no alcohol, alcohol w/ AEC or alkaline phosphate rxn will dissolve product & give false-negative |
| AEC | 3-amino-9-ethylcarbazole, chromogen that gives brick red color rxn w/ peroxidase in presence of H2O2 |
| Frozen sections & immunofluorescence | Classic prep unfixed tissue, antigenic reactivity least impaired, fluorescent antibody staining strongest, soluble antigens lost |
| Formalin-fixed paraffin & immunofluorescence | Rarely used b/c inconsistent results, antigenic reactivity damaged, formaldehyde cross-linking means epitopes unavailable |
| Vimentin | To determine if tissue is over-fixed, usually excellent staining on paraffin |
| B5 fixation | Great for demo of intracytoplasmic antigens, more intense staining, bad for demo of surface membrane immunoglobulins |
| Glutaraldehyde fixation | Best morphological preservation, irreversibly blocks antigenic determinants |
| Most important for IHC fixation: | Standardize fixation, if possible in formalin no more than 24 hrs |
| Epitope enhancement | aka antigen retrieval, improve detectability of antigens in formalin-fixed tissue, can further dilute antibodies, expose blocked epitopes, more intense rxn w/ decr'd incubation times, more uniform staining, consistent stains, better standardization |
| 2 methods of epitope enhancement | Heat-induced epitope retrieval (HIER), enzyme-induced epitope retrieval (EIER) - older less used |
| HIER methods: | Microwave, steamer, pressure cooker, retrieval soln. - TRIS-HCl or sodium acetate buffer, pH 8 to 9, some methods cause morphological damage |
| EIER methods: | Use proteolytic enzymes, digestion 1-60 min. depending, may red. nonspecific staining, can incr. if not careful, may weaken specific staining, HIER better for most antibodies |
| Positive control | Run w/ each antibody every stain, use commercially prep'd controls only to check reagents, prepare same as diagnostic slide |
| Negative control (specific) | Tissue expected negative by antibody, prepare same as diagnostic, same antibody as diagnostic |
| Negative control (nonspecific) | Prepare same as diagnostic, same tissue as diagnostic, antibody not specific for antigen of interest |
| Evaluation of new antibodies includes: | w/ no retrieval, w/ HIER, w/ EIER, w/ HIER & EIER, determine which is best, determine optimal dilution |
| To make 1mL of 1:x dilution: | 1,000/x = µL raw antisera req'd 1,000 - 1,000/x = µL antibody dilution buffer req'd |
| Antibody storage: | Polypropylene, polycarbonate, or borosilicate glass, may add 0.1 to 1.0% BSA to red. loss thru polymerization & absorption into container, 4°C to 8°C, may store aliquot dilutions at -70°C |
| Blocking reactions: | H2O2 w/ methanol blocks endogenous peroxidase activity, esp. if many rbc, Casein can block, enhances sensitivity innocuous protein soln. (nonimmune serum of same species as 2° antibody) before antibody, binds charged sites, decr. nonspecific staining, |
| Multilink secondary antibodies: | Mix of biotinylated anti-mouse, anti-rabbit, & other species antibodies, don't have to keep stock of as many specific antibodies |
| DAB reaction product intensification may be done w/: | NiCl, CuCl, CoCl - may incr. background, can't use w/ some procedures, imidazole - pH 7.6, better than metals, inhibits hemoglobin pseudoperoxidase activity OsO4 - after DAB rxn, may darken background |
| Problem - specimen & positive control unstained, possible causes 1: | 1° antibody ommitted substrate-chromagen improperly made, reagents used wrong order, alcohol-based counterstain, mounting medium used w/ AEC, fast red, or tetrazolium - use water-based, sections dried during procedure |
| Problem - specimen & positive control unstained, possible causes 2: | Wrong 2°, omitted labelled reagent, sodium azide contamination in buffer bath |
| Problem: specimen & positive control have weak staining, possible causes: | Substrate-chromagen prep'd wrong, 1° antibody too dilute, defective, insufficient incubation time, defective reagent(s), too much rinse buffer on slides, epitope enhancement done incorrectly |
| Problem: specimen weak staining, positive control stained, possible causes: | Low conc. antigen or masked during fixation |
| Problem: specimen & positive control have excessive background staining, possible causes: | Paraffin present, endogenous products - check blocking reagent, excessive adhesive, slides not well-washed w/ buffer, high conc. 1° antibody, link or label reagent, 1° antibody or substrate incubation too long |
| Problem: specimen excessive background, positive control no background, possible causes: | Free antigen b/c necrosis, autolysis, degeneration - interpret in areas of less intense background |
| Basic PAP, ABC Immunoperoxidase, Immunoperoxidase w/ 3-step indirect method, & LAB purpose | Localization of tissue antigens |
| Basic PAP Immunoperoxidase facts | Fixation: B5, Zenker, Bouin probably won't need epitope enhancement, formalin better some antigens |
| Basic PAP Immunoperoxidase reagents | 1° antibodies PBS linking serum PAP AEC Mayer hematoxylin aq. mounting medium or crystal mount |
| Basic PAP, ABC Immunoperoxidase, Immunoperoxidase w/ 3-step indirect method, & LAB results | Positive rxn - brick red |
| Modified PBS buffer stock solution | potassium phosphate, dibasic sodium phosphate, monobasic NaCl |
| AEC solution | 3-amino-9ethylcarbazole N-N dimethyl formamide acetate buffer H2O2 |
| Acetate buffer for IHC staining solution | sodium acetate, anhydrous or trihydrate acetic acid |
| ABC-Immunoperoxidase & LAB facts | Fixative: 10% NBF, zinc formalin, B5, Zenker, Bouin |
| ABC-Immunoperoxidase reagents | 1° antibodies w/ BSA PBS biotinylated antibodies Vectastain elite ABC reagent AEC Mayer hematoxylin aq. mounting medium or Crystal Mount |
| BSA | Bovine serum albumin, protein additive for antibody dilutions, doesn't react w/ other proteins, incr. protein conc. of soln. to red. polymerization, can use as non-specific blocking reagent |
| LAB reagents | 1° antibodies w/ BSA in PBS PBS biotinylated goat anti-mouse Ig avidin-D w/ horseradish peroxidase AEC (develop) Mayer hematoxylin |
| Avidin-D conjugated w/ Horseradish Peroxidase solution | PBS, pH 7.4 stock avidin horseradish peroxidase normal human serum |
| LAB technique excellent w/: | Alkaline phosphatase streptavidin, when using alkaline phosphatase label substrate should contain napthol phosphate & chromogen |
| Biotin-Avidin-Horseradish Peroxidase purpose | Primarily surface marking of lymphomas |
| Biotin-Avidin-Horseradish Peroxidase facts | Frozen sections - fix briefly in acetone, dry, fix in acetone again, Sections: cryostat 2-3 µm |
| Biotin-Avidin-Horseradish Peroxidase reagents | monoclonal antibodies w/ BSA PBS biotinylated goat anti-mouse Ig w/ thimerosal avidin-D w/ horseradish peroxidase w/ thimersol DAB copper sulfate methylene blue (or hematoxylin) |
| Biotin-Avidin-Horseradish Peroxidase results | Surface antibody demo'd as brown rings |
| DAB solution for Biotin-Avidin-Horseradish Peroxidase | DAB PBS 30% H2O2 |
| DAB | 3,3'-diaminobenzidine, chromogen - brownish-red precipitate w/ peroxidase in presence of H2O2, water-soluble |
| Copper Sulfate solution for Biotin-Avidin-Horseradish Peroxidase | CuSO4 NaCl dH2O |
| Immunoperoxidase staining w/ 3-step indirect method facts | Frozen sections - fix 10 min. acetone, may air-dry, 2-3 µm, cytospin preps - don't fix, just dry |
| Immunoperoxidase staining w/ 3-step indirect method reagents | monoclonal antibodies w/ TRIS & BSA TRIS-NaCl wash buffer soln. peroxidase-conjug'd rabbit anti-mouse Ig peroxidase-conjug'd goat anti-rabbit Ig AEC Mayer hematoxylin glycerin jelly or prep for Crystal Mount |
| TRIS-sodium chloride wash buffer solution | NaCl 0.9% TRIS base dH2O pH 7.4 w/ HCl |
| BSA 0.1% for Immunoperoxidase staining w/ 3-step indirect method solution | BSA 22% TRIS-NaCl wash buffer |
| Basic IHC staining steps: | 1° antibody binds sites of interest, 2° antibody binds/reacts w/ 1° , 2° antibody attaches to enzyme molecule system, enzyme system forms precipitate when exposed to substrate & chromagen, counterstain nuclei to show background |
| What slides are best for basic PAP, ABC immunoperoxidase, & LAB? | poly-L-lysine coated or silanized slides |
| CK 20 | To ID colon cancer |
| CD3 | To ID T cell lymphomas |
| CD20 | To ID B cell lymphomas |
| HMB45 | To ID melanomas |
| Her2 | To ID breast carcinomas |
| GFAP | To ID glioblastomas |
| AE1/AE3 | Cytokeratin antibody to ID carcinomas |
| Enzymes used in EIER: | Trypsin w/ CaCl (or PBS), 10 min, pepsin in HCl, ficin, cytokeratin 30-60 min., pronase in TRIS buffer, protease in PBS |
| When possible better to use DAB than AEC because: | w/ DAB sections can be dehydrated, cleared, mounting w/ resinous medium sections have greater clarity & are permanent, can use in basic PAP, ABC, LAB, & 3-step indirect methods |
| Alkaline phosphatase chromogens | Fast red TR, fast red-violet LB, these chromogens can't be dehydrated & cleared, will break down w/ prolonged dehydration |
| Coenzyme | Organic compound that works w/ enzyme to influence rate of rxn, may be vitamins, may act as cosubstrate, may receive what is removed from substrate |
| Cofactor | Chemicals that speed action of enzyme, complex proteins or simple metallic ions, may receive what is removed from substrate |
| ISH purpose | in situ hybridization, to detect nucleotide sequence of interest, usu. detect specific mRNAs |
| ISH facts | Best on frozen, fixed sections, paraformaldehyde preferred, store fixed, cryoprotected @ -80°C, store fixed, immersed in methanol @ -20°C |
| ISH probes | Nucleotide sequence complimentary to sequence of interest, labeled, single strand, 50-300 nucleotides, may be difficult obtain appropriate probe, create thru cloning |
| Isotopic hybridization | Uses probe labeled w/ radioactive isotopes |
| Non-isotopic hybridization | Uses probe conjugated w/ non-radioactive isotopes, usu. biotinylated, fluorescent molecules, digoxigenin, bromodeoxyuridine |
| ISH steps | Tissue incubated w/ probe, target hybridization, signal amplification (opt. - depends on label type, may decr. background) visualization (how depends on label type) |
| FISH | Fluorescent in situ hybridization, can visualize multiple targets simultaneously |
| CISH | Chromogenic in situ hybridization, uses bright-field microscopy, can view target & morphology simultaneously |
| S100 | Calcium-binding antibody, Langerhans cells (skin), interdigitating reticulum cells (paracortex of lymph nodes) |
| PSA | Prostate specific antigen, detect prostatic carcinoma |
| CEA | Carcino embryonic antigen, detect intestinal carcinomas, other tumors, differentiate tumors, origins |
| What fixative & length of time are recommended by ASCO/CAP for breast tissue for HER2/neu testing? | 10% NBF, min. 6 hrs., max 48 hrs. |
| Pan-cytokeratin antibody | |
| LCA/CD45 | Stains cell membrane of leukyocytes |
| Kappa antibody | Stains kappa light chain, useful to ID leukemias, plasmacytomas, & some non-Hodgkin lymphomas |
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