Staining
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| Antigen | immunogen,
induces immune response,
proteins best, may be polysaccharides, nucleic acids, polymers,
bacteria & viruses most common
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| 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)
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| 5 classes of antibodies | Immunoglobulin G (IgG) γ
Immunoglobulin A (IgA) α
Immunoglobulin M (IgM) μ
Immunoglobulin D (IgD) Δ
Immunoglobulin E (IgE) ε
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| Epitope | Region of antigen antibodies bind,
many antibodies can bind same antigen,
each antibody binds one epitope
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| Polyclonal antibody | Pool of antibodies from many clones of lymphocytes
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| Monoclonal antibodies | Primarily made in mice,
so link usu. anti-mouse,
high homogeneity,
nonspecific antibodies absent,
little batch-to-batch variability
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| 4 methods of IHC staining | Direct, indirect,
unlabeled or soluble enzyme immune complex,
avidin-biotin
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| Direct method | Known, labeled antibody used to ID tissue antigens
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| What can be used to label an antibody? | Fluorescein isothiocyanate (FITC), fluorescent dye,
enzymes (horseradish peroxidase, alkaline phosphatase, glucose oxidase) - react w/ chromagen
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| Indirect method | Serum added to soln. w/ known antigens,
labeled antibody used to detect bound antibodies from serum
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| 2-step indirect method | Technically direct,
2° labeled antibody used to localize 1° unlabeled defined antibody
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| 3-step indirect method | 2nd & 3rd antibodies labeled w/ enzymes,
good to incr. staining intensity
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| 2- & 3-step indirect methods are usually used w/: | Enzyme-conjugated antibodies for light microscopy,
multi-step indirect enhances tissue staining for light microscopy
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| 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
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| 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)
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| Avidin | High affinity for biotin,
originally from eggwhites, now use streptavidin from Streptomyces avidinii
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| Immunofluorescence staining | Most common FITC & rhodamine,
when fluorochrome attached to antibody rxn sites easily visualized
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| Best hematoxylin counterstain for IHC? | Mayer b/c no alcohol,
alcohol w/ AEC or alkaline phosphate rxn will dissolve product & give false-negative
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| AEC | 3-amino-9-ethylcarbazole,
chromogen that gives brick red color rxn w/ peroxidase in presence of H2O2
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| Frozen sections & immunofluorescence | Classic prep unfixed tissue,
antigenic reactivity least impaired,
fluorescent antibody staining strongest,
soluble antigens lost
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| Formalin-fixed paraffin & immunofluorescence | Rarely used b/c inconsistent results,
antigenic reactivity damaged,
formaldehyde cross-linking means epitopes unavailable
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| Vimentin | To determine if tissue is over-fixed,
usually excellent staining on paraffin
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| B5 fixation | Great for demo of intracytoplasmic antigens,
more intense staining,
bad for demo of surface membrane immunoglobulins
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| Glutaraldehyde fixation | Best morphological preservation,
irreversibly blocks antigenic determinants
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| Most important for IHC fixation: | Standardize fixation,
if possible in formalin no more than 24 hrs
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| 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
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| 2 methods of epitope enhancement | Heat-induced epitope retrieval (HIER),
enzyme-induced epitope retrieval (EIER) - older less used
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| HIER methods: | Microwave, steamer, pressure cooker,
retrieval soln. - TRIS-HCl or sodium acetate buffer,
pH 8 to 9,
some methods cause morphological damage
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| 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
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| Positive control | Run w/ each antibody every stain,
use commercially prep'd controls only to check reagents,
prepare same as diagnostic slide
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| Negative control (specific) | Tissue expected negative by antibody,
prepare same as diagnostic,
same antibody as diagnostic
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| Negative control (nonspecific) | Prepare same as diagnostic,
same tissue as diagnostic,
antibody not specific for antigen of interest
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| Evaluation of new antibodies includes: | w/ no retrieval,
w/ HIER,
w/ EIER,
w/ HIER & EIER,
determine which is best,
determine optimal dilution
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| 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
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| 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
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| 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,
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| Multilink secondary antibodies: | Mix of biotinylated anti-mouse, anti-rabbit, & other species antibodies,
don't have to keep stock of as many specific antibodies
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| 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
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| 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
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| Problem - specimen & positive control unstained, possible causes 2: | Wrong 2°,
omitted labelled reagent,
sodium azide contamination in buffer bath
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| 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
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| Problem: specimen weak staining, positive control stained, possible causes: | Low conc. antigen or masked during fixation
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| 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
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| Problem: specimen excessive background, positive control no background, possible causes: | Free antigen b/c necrosis, autolysis, degeneration - interpret in areas of less intense background
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| Basic PAP, ABC Immunoperoxidase, Immunoperoxidase w/ 3-step indirect method, & LAB purpose | Localization of tissue antigens
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| Basic PAP Immunoperoxidase facts | Fixation: B5, Zenker, Bouin probably won't need epitope enhancement,
formalin better some antigens
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| Basic PAP Immunoperoxidase reagents | 1° antibodies
PBS
linking serum
PAP
AEC
Mayer hematoxylin
aq. mounting medium or crystal mount
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| Basic PAP, ABC Immunoperoxidase, Immunoperoxidase w/ 3-step indirect method, & LAB results | Positive rxn - brick red
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| Modified PBS buffer stock solution | potassium phosphate, dibasic
sodium phosphate, monobasic
NaCl
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| AEC solution | 3-amino-9ethylcarbazole
N-N dimethyl formamide
acetate buffer
H2O2
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| Acetate buffer for IHC staining solution | sodium acetate, anhydrous or trihydrate
acetic acid
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| ABC-Immunoperoxidase & LAB facts | Fixative: 10% NBF, zinc formalin, B5, Zenker, Bouin
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| ABC-Immunoperoxidase reagents | 1° antibodies w/ BSA
PBS
biotinylated antibodies
Vectastain elite ABC reagent
AEC
Mayer hematoxylin
aq. mounting medium or Crystal Mount
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| 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
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| LAB reagents | 1° antibodies w/ BSA in PBS
PBS
biotinylated goat anti-mouse Ig
avidin-D w/ horseradish peroxidase
AEC (develop)
Mayer hematoxylin
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| Avidin-D conjugated w/ Horseradish Peroxidase solution | PBS, pH 7.4
stock avidin horseradish peroxidase
normal human serum
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| LAB technique excellent w/: | Alkaline phosphatase streptavidin,
when using alkaline phosphatase label substrate should contain napthol phosphate & chromogen
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| Biotin-Avidin-Horseradish Peroxidase purpose | Primarily surface marking of lymphomas
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| Biotin-Avidin-Horseradish Peroxidase facts | Frozen sections - fix briefly in acetone, dry, fix in acetone again,
Sections: cryostat 2-3 µm
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| 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)
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| Biotin-Avidin-Horseradish Peroxidase results | Surface antibody demo'd as brown rings
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| DAB solution for Biotin-Avidin-Horseradish Peroxidase | DAB
PBS
30% H2O2
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| DAB | 3,3'-diaminobenzidine,
chromogen - brownish-red precipitate w/ peroxidase in presence of H2O2,
water-soluble
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| Copper Sulfate solution for Biotin-Avidin-Horseradish Peroxidase | CuSO4
NaCl
dH2O
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| 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
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| 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
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| TRIS-sodium chloride wash buffer solution | NaCl 0.9%
TRIS base
dH2O
pH 7.4 w/ HCl
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| BSA 0.1% for Immunoperoxidase staining w/ 3-step indirect method solution | BSA 22%
TRIS-NaCl wash buffer
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| 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
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| What slides are best for basic PAP, ABC immunoperoxidase, & LAB? | poly-L-lysine coated or silanized slides
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| CK 20 | To ID colon cancer
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| CD3 | To ID T cell lymphomas
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| CD20 | To ID B cell lymphomas
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| HMB45 | To ID melanomas
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| Her2 | To ID breast carcinomas
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| GFAP | To ID glioblastomas
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| AE1/AE3 | Cytokeratin antibody to ID carcinomas
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| 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
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| 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
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| Alkaline phosphatase chromogens | Fast red TR, fast red-violet LB,
these chromogens can't be dehydrated & cleared,
will break down w/ prolonged dehydration
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| 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
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| Cofactor | Chemicals that speed action of enzyme,
complex proteins or simple metallic ions,
may receive what is removed from substrate
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| ISH purpose | in situ hybridization,
to detect nucleotide sequence of interest,
usu. detect specific mRNAs
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| ISH facts | Best on frozen, fixed sections,
paraformaldehyde preferred,
store fixed, cryoprotected @ -80°C,
store fixed, immersed in methanol @ -20°C
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| ISH probes | Nucleotide sequence complimentary to sequence of interest,
labeled, single strand, 50-300 nucleotides,
may be difficult obtain appropriate probe,
create thru cloning
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| Isotopic hybridization | Uses probe labeled w/ radioactive isotopes
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| Non-isotopic hybridization | Uses probe conjugated w/ non-radioactive isotopes,
usu. biotinylated, fluorescent molecules, digoxigenin, bromodeoxyuridine
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| ISH steps | Tissue incubated w/ probe,
target hybridization,
signal amplification (opt. - depends on label type, may decr. background)
visualization (how depends on label type)
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| FISH | Fluorescent in situ hybridization,
can visualize multiple targets simultaneously
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| CISH | Chromogenic in situ hybridization,
uses bright-field microscopy,
can view target & morphology simultaneously
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| S100 | Calcium-binding antibody,
Langerhans cells (skin),
interdigitating reticulum cells (paracortex of lymph nodes)
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| PSA | Prostate specific antigen,
detect prostatic carcinoma
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| CEA | Carcino embryonic antigen,
detect intestinal carcinomas, other tumors,
differentiate tumors, origins
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| 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.
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| Pan-cytokeratin antibody |
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| LCA/CD45 | Stains cell membrane of leukyocytes
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| Kappa antibody | Stains kappa light chain,
useful to ID leukemias, plasmacytomas, & some non-Hodgkin lymphomas
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