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IHC

Staining

QuestionAnswer
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
Created by: CCF