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29 Cards in this Set

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