Staining
Quiz yourself by thinking what should be in
each of the black spaces below before clicking
on it to display the answer.
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| show | immunogen,
induces immune response,
proteins best, may be polysaccharides, nucleic acids, polymers,
bacteria & viruses most common
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| show | 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 | show 🗑
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| Epitope | show 🗑
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| Polyclonal antibody | show 🗑
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| show | Primarily made in mice,
so link usu. anti-mouse,
high homogeneity,
nonspecific antibodies absent,
little batch-to-batch variability
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| show | Direct, indirect,
unlabeled or soluble enzyme immune complex,
avidin-biotin
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| Direct method | show 🗑
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| show | Fluorescein isothiocyanate (FITC), fluorescent dye,
enzymes (horseradish peroxidase, alkaline phosphatase, glucose oxidase) - react w/ chromagen
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| show | Serum added to soln. w/ known antigens,
labeled antibody used to detect bound antibodies from serum
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| show | Technically direct,
2° labeled antibody used to localize 1° unlabeled defined antibody
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| show | 2nd & 3rd antibodies labeled w/ enzymes,
good to incr. staining intensity
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| show | Enzyme-conjugated antibodies for light microscopy,
multi-step indirect enhances tissue staining for light microscopy
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| show | 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: | show 🗑
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| show | High affinity for biotin,
originally from eggwhites, now use streptavidin from Streptomyces avidinii
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| show | Most common FITC & rhodamine,
when fluorochrome attached to antibody rxn sites easily visualized
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| show | Mayer b/c no alcohol,
alcohol w/ AEC or alkaline phosphate rxn will dissolve product & give false-negative
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| AEC | show 🗑
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| show | Classic prep unfixed tissue,
antigenic reactivity least impaired,
fluorescent antibody staining strongest,
soluble antigens lost
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| Formalin-fixed paraffin & immunofluorescence | show 🗑
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| show | To determine if tissue is over-fixed,
usually excellent staining on paraffin
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| show | Great for demo of intracytoplasmic antigens,
more intense staining,
bad for demo of surface membrane immunoglobulins
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| Glutaraldehyde fixation | show 🗑
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| show | Standardize fixation,
if possible in formalin no more than 24 hrs
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| Epitope enhancement | show 🗑
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| 2 methods of epitope enhancement | show 🗑
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| show | 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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| show | 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 | show 🗑
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| Negative control (specific) | show 🗑
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| show | Prepare same as diagnostic,
same tissue as diagnostic,
antibody not specific for antigen of interest
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| show | 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: | show 🗑
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| show | 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: | show 🗑
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| Multilink secondary antibodies: | show 🗑
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| DAB reaction product intensification may be done w/: | show 🗑
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| show | 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: | show 🗑
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| Problem: specimen & positive control have weak staining, possible causes: | show 🗑
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| show | Low conc. antigen or masked during fixation
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| show | 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: | show 🗑
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| show | Localization of tissue antigens
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| show | Fixation: B5, Zenker, Bouin probably won't need epitope enhancement,
formalin better some antigens
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| show | 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 | show 🗑
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| Modified PBS buffer stock solution | show 🗑
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| AEC solution | show 🗑
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| Acetate buffer for IHC staining solution | show 🗑
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| show | Fixative: 10% NBF, zinc formalin, B5, Zenker, Bouin
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| show | 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 | show 🗑
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| LAB reagents | show 🗑
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| show | PBS, pH 7.4
stock avidin horseradish peroxidase
normal human serum
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| show | Alkaline phosphatase streptavidin,
when using alkaline phosphatase label substrate should contain napthol phosphate & chromogen
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| show | Primarily surface marking of lymphomas
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| show | 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 | show 🗑
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| show | Surface antibody demo'd as brown rings
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| DAB solution for Biotin-Avidin-Horseradish Peroxidase | show 🗑
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| DAB | show 🗑
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| Copper Sulfate solution for Biotin-Avidin-Horseradish Peroxidase | show 🗑
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| Immunoperoxidase staining w/ 3-step indirect method facts | show 🗑
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| Immunoperoxidase staining w/ 3-step indirect method reagents | show 🗑
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| show | NaCl 0.9%
TRIS base
dH2O
pH 7.4 w/ HCl
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| show | BSA 22%
TRIS-NaCl wash buffer
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| Basic IHC staining steps: | show 🗑
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| What slides are best for basic PAP, ABC immunoperoxidase, & LAB? | show 🗑
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| show | To ID colon cancer
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| CD3 | show 🗑
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| show | To ID B cell lymphomas
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| show | To ID melanomas
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| Her2 | show 🗑
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| GFAP | show 🗑
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| AE1/AE3 | show 🗑
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| show | 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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| show | 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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| show | Fast red TR, fast red-violet LB,
these chromogens can't be dehydrated & cleared,
will break down w/ prolonged dehydration
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| show | 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 | show 🗑
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| ISH purpose | show 🗑
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| show | 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 | show 🗑
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| show | Uses probe labeled w/ radioactive isotopes
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| show | Uses probe conjugated w/ non-radioactive isotopes,
usu. biotinylated, fluorescent molecules, digoxigenin, bromodeoxyuridine
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| ISH steps | show 🗑
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| FISH | show 🗑
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| show | Chromogenic in situ hybridization,
uses bright-field microscopy,
can view target & morphology simultaneously
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| show | Calcium-binding antibody,
Langerhans cells (skin),
interdigitating reticulum cells (paracortex of lymph nodes)
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| PSA | show 🗑
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| CEA | show 🗑
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| What fixative & length of time are recommended by ASCO/CAP for breast tissue for HER2/neu testing? | show 🗑
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| show |
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| LCA/CD45 | show 🗑
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| show | Stains kappa light chain,
useful to ID leukemias, plasmacytomas, & some non-Hodgkin lymphomas
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