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31 Cards in this Set
- Front
- Back
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Steps for tissue preparation
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1. Fixation
2. Dehydration 3. Clearing 4. embedding 5. Sectioning 6. Staining |
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Fixation
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Crosslink and immobilize molecules in cell
takes about 12 hours |
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Formaldehyde
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The fixative used in light microscopy
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Buffered Gluteraldehyde
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used as the fixative for electron microscopy
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Osmium Tetroxide
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stains and preserves lipids and proteins
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Lead
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stains proteins
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Dehydration
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graded ethanol solutions to replace tissue water with organic solvents
6-24 hours |
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Clearing
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left in benzene, xylene or toluene so as to be miscible with parrafin
1-6 hrs |
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Embedding
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dip in paraffin so it goes into all intracellular spaces
1-3hrs |
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Sectioning
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with a microtome and steel for paraffin and glass for plastic resin cut into very slin sections
cryostat (freezing microtome) preserves lipids |
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Staining
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deparaffined with xylol and rehydrated to take up stain
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Condensor
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glass lens that focuses light on the specimen in a light microscope
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objective lens
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what the lens focuses the light with and produces a magnified specimen, most important
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basophillic dyes
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stain chromatin nuclei and RER because of high concentration of negative charges
stain blue example is hemotoxylin |
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acidic dyes
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stain mitochondria, secretory granules and collagen
stains red example eosin |
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H&E
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heotoxylin and eosin, cytoplasm tends to be pink and nucleus blue
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Elastic Stains
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stain elastin
stain a dark brown |
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Silver Stains
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stains neuron cell body and neurofillaments in axons as well as Type III collagen black
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colloidal carbon or Toluidine blue.
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a vital dye that identifies phagocytic cells because will inject invivo and thes cells will pick up the die
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PAS
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reveals glycogen which is prominent in liver and muscle
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Direct Fluorescent staining
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Tissues incubated directly with fluorescent or peroxidase labeled goat antibody to human protein X.
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Indirect Fluorescent staining
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Tissues first incubated with unlabeled goat antibody to human protein X, then incubated with a labeled rabbit anti-antibody to the goat antibody which binds to many more sites producing a much brighter staining
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Peroxidase
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produces a dark electron dense precipitate when antibody incubated with hydrogen peroxide and DAB
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Confocal microscope
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allows to do depth sectioning or take image in focus at various depths and then whole image is put together by computer
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Multiphoton Microscope
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Long wavelength excitation pulses of lasers are given to the specimen over very short periods of time. Fluorescence is generated only at the focal spot with no out-of-focus light to reject as has to be done in confocal microscopy.
CAN WATCH IN VIVO |
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TEM
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fix with glutaraldehyde for proteins and osmium tetroxide for proteins and lipids; 50-100nm specimen; lead for staining proteins
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SEM
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fix, dry, coat with platinum, then carbon.
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Freeze Fracture
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freeze
then fracture along plane of least molecular binding (ex bewteen hydrophobic tails of E and P phospholipids) look at with TEM |
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Freeze Etch
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frozen at a lower temperature and allow for some water sublimation while temp goes up
see more detail this way look at with TEM |
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Autoradiography Steps
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1 living cells exposed to a paritcular radioactive dye and allowed to take up
2. washed to remove any extra dye, then fixed and sectioned 3. placed on slides 4. dipped and produces a mono layer of silver bromide in gelatin 5. stored and silver bromide act as radioactivity detectors 6. exposed like film and radioactivity is shown by where can see dark silver bromide granules |
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tritium
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used to study metabolism
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