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29 Cards in this Set
- Front
- Back
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4 main light microscopy applications for clinical path:
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-H and E
-Cyto/histochemistry -Immunohistochemistry -Autoradiography |
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L=4 Main microscopy types used fo rresearch:
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-Autoradiography
-insitu hybridization -Confocal microscopy -Electron microscopy |
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3 types of EM applications:
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-Freeze fracture
-EM Immunohistochemistry -Scanning EM |
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Steps in Tissue Prep:
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1. Fixation (formaldehyde)
2. Dehydrate (alcohol) 3. Clear (emb miscible solvent) 4. Embed (plastic/parafin) 5. Section (microtome) 6. Stain |
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How they stain:
-nuclei -cytoplasm |
nuclei = basophilic
cytoplasm = acidophilic |
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Eosin:
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-Acidic/electron donating dye
-Stains acidophilic, positively charged things (cytoplasm) |
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Hematoxylin
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Basic dye - has pos charges to accept electrons
-Basophilic things - nucleic acids are neg charged and like the basic dyes. |
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what is detected with Schiff reagent?
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-DNA
-Glycogen |
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What is detected with Sudan dyes?
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Lipids
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Objective of histochemistry:
-example |
to detect a specific protein by its enzymatic activity
Ex: succinic dehydrogenase - marks red muscle fibers mitochondria |
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Objective of immunohistochemistry:
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To detect specific proteins by antigen reactions.
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Most common type of antibody used in imunohistochemistry:
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Secondary - 2nd Ab has a label and reacts w/ the 1st Ab that reacts with the target Ag
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objective of autoradiography:
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To detect a specific cellular activity - i.e. DNA synthesis during S phase, or protein synthesis
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What is commonly used to label nuclei during DNA synthesis?
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H-TdR - tritiated thymidine
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What develops in cells during DnA syntheis when labeled with H-TdR?
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Hotspots - places where B-electron emissions reacted with silver bromide in a film that was coated on cells.
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Where do intestinal stem cells divide? Then where do they go?
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In intestinal Crypts - then they go to the top of villi to do their job.
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what technique showed that intestinal cells divide in crypts?
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H-TdR
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What exactly do hotspots look like?
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Black silver grains over nuclei
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objective of FISH:
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to detect specific RNA (or DNA) with labeled probes
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What is the objective of confocal microscopy?
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To detect high resolution 3D images in thick sections
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How does confocal imaging work?
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Flourescent probes are used to label macromolecules, then excited with a laser, then light from a cross-section is focused on a detector to create a confocal image.
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WHat is the objective of EM?
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High-resolution, high magnificn look at the ULTRASTRUCTURE of cells.
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How does the resolution of EM compare to light microscopy?
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EM = 0.2 nm
LM = 1 um |
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What molecules are used to enhance EM?
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-Osmium stain
-Lead/uranium salts (electron dense to provide contrast) |
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What type of imaging is freeze fracture? What is it used for?
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Special type of EM - used to study membrane structures.
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Where does the fracture occur in freeze fracture?
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Between the hydrophobic layers of opposing leaflets in the phospholipid bilayer.
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What is the Eface? Pface?
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E = extracellular = outside face
P = cytoplasmic = intracellular |
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What can be achieved by Freeze fracture?
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A look at the INTEGRAL membrane proteins
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What is the objective of scanning EM?
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To obtain a 3D surface image
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