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12 Cards in this Set
- Front
- Back
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Light MicroscopyFixation |
1. Fix sample with an aqueous formaldehyde solution 2. Dehydrate by soaking in a series of ethanol solutions of increasing concentration (50%->70%->90%->100%) 3. Embed the dehydrated tissue in paraffin wax |
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Light MicroscopyResolution |
= ~0.2μm or 200 nm No resolution allows light microscopy to resolve a biological membrane |
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Electron MicroscopyResolution |
=~1.0 nm in biological samples (remember 1Å = 0.1nm) At 8-10nm you can resolve a biological membrane |
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Electron MicroscopyFixation |
1. Fix sample in glutaraldehyde then 〖OsO〗_4 (osmium tetroxide) 2. Dehydrate with increasing concentration of ethanol solution as in light microscopy 3. Embed in hard plastic (ex. Epoxy or acrylate resins) Allows thinner sections to be cut than in light microscopy |
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Hematoxylin & Eosin Staining (H&E) |
General Tissue staining for LM For staining of paraffin section Hematoxylin A basic dye - has a positive charge (cationic) Binds negatively charged (anionic) tissue components (components that exhibit basophilia) Eosin An acid dye - has a negative charge and binds positively charged tissue components These positively charged components are said to exhibit acidophilia |
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Periodic Acid Schiff (PAS) |
Selective Tissue Stains for LM A reaction that stains carbohydrates on cell molecules Ex. Glycoproteins, glycogen, etc. |
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Toluidine Blue |
Selective Tissue Stains for LM A metachromatic dye - changes color depending on the amount of dye that binds a molecule Ex. Stains nucleic acids blue while it stains polysaccharides purple A basic dye with a high affinity for anionic/acidic tissue components |
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Direct Immunocytochemistry |
Specific Tissue Stains for LM A labeled primary antibody directly binds the target antigen within the tissue sample Specific Tissue Stains for LM The labeled structures are then observed in a fluorescence microscope where light of a specific wavelengths excites the fluorochrome -> emission o fa longer wavelength of light This emitted light can be viewed by the eye or captured on a camera or photomultiplier tube Not useful when there is a low concentration of the target as the emitted light may be too weak to view (only 1 primary antibody can bind each antigen) However it is easier to do than indirect immunochemistry |
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Indirect Immunocytochemistry |
Specific Tissue Stains for LM An unlabeled primary antibody binds directly with the target antigen in the tissue A labeled secondary antibody then binds the primary antibody -> amplification of the fluorescent signal (because multiple secondary antibodies can bind a single primary antibody) |
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Immunoperoxidase Immunocytochemistry |
Specific Tissue Stains for LM An unlabeled primary antibody binds directly with the target tissue A secondary antibody tagged with a peroxidase enzyme then binds the primary antibody The addition of the enzyme substrate activates the enzyme -> substrate cleavage -> a fluorescent compound This resultant fluorescent compound is insoluble and precipitates at the site of cleavage -> fluorescence in the region of the target |
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Intrinsically Fluorescent Molecules |
Molecules with autoflourescence Useful for tracking target substrate movement in living cells DAPI - for tagging nucleic acids and fluoresces blue Can be introduced into a cell in order to visualize DNA during mitosis GFP - for tagging proteins Must be transfected into the target via an expression vector that encodes a GFP-target protein chimera |
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Immunocytochemistry for EM |
Immunogold An antibody with an attached colloidal gold molecule binds the target -> electron dense markers on the targets surface Heavy Metal An antibody with an attach ferritin or other heavy metal (ex. Osmium) binds the target -> electron dense markers on target |
