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Flashcards in Lecture 1 Deck (12):

Preparation For Light Microscopy

1. Fixation- Formalin (formaldehyde) which reacts with tissues to cross link proteins and other substances
2. Dehydration- Alcohol or acetone
3. Embedding- paraffin or plastic
4. Sectioning- generally 2-10 um thick
5. Mounting- glass slide


Hematoxylin and eosin (H and E)

H has positive charge and stains negatively charged structures (nucleus). E is negatively charged and stains positively charged substances (cytoplasm).


Periodic Acid Schiff Stain

For carbohydrates like sugars (glycogen and glycoproteins)


Aldehyde Fuchsin

Stains Elastic Fibers and b cells of the pancreatic islets. Connective tissue



Stains collagen fibers


Silver Stain

Stains golgi apparatus and reticular fibers


Resolution of Light Microscopy

- about 0.2 microns.
- useful Magnification of about 1,500 times


Resolution of Transmission Electron Microscope

- about 1-1.45 nanometer
- useful magnification greater than 500,000 times


Resolution of Scanning Electron Microscope

- about 2 nm
- useful magnification is greater than x100,000


Preparation for TEM

1. Fixation - with glutaraldehyde that fixes proteins and nucleic acids followed by osmium tetroxide that fixes lipids.
2. Dehydration
3. Embeddign: plastic such as Epon
4. Sectioning: 20-100 nm thick using ultramicrotome using glass or diamond knife
5. Mounting: wire mesh
6. Staining: heavy metals are used to enhance electron density


Freeze Fracture

- method of specimen preparation
- follows the hydrophobic region of the membrane exposing 2 fracture faces
- the E face- inwardly facing outer half
- P face- the outwardly facing inner half


Preparation of Scanning Electron Microscopy

1. Fixation and dehydration - same as TEM
2. Dried using the critical point method to avoid drying artifacts
3. mounting - glued to a metal stub
4. coated with metal (gold/palladium) so it will enhance emission of electrons