Semi-final I Histological Specimen Prep Methods

Question 2

What are the steps of normal light microscope slide prep?

Answer 2

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1. Fixation - specimen immersed in or injected with aldehyde to denature autolytic enzymes
2. **Dehydration/Clearing - **ascending conc. of alcohol to dehydrate, hydrophobe (Xylene) to finish
3. **Embedding - **immersed in paraffin wax
4. **Sectioning **- **3-5 µm sections cut w/ microtome
5. **Deparaffinization, Rehydration - **par. wax evaporated, descending conc. of alcohol to rehydrate, immersed in phosphate-buffered saline
6. Staining - many possible stains, HE most common
7. **Mounting/Cover Slip ** - redehydrated, mounting media (synth. resin, etc.) added and covered with coverslip

Question 3

What are the 2 main reasons for tissue prep in light microscopy?

Answer 3

1. using thin enough sections for light to penetrate
2. staining to allow visualization and differentiation of structures

Question 4

What are the two components of HE staining? How do they differ?

Answer 4

1. **Hematoxilin **- basic, binds to acidic structures such as nucleic acids (within DNA)
2. Eosin - acidic, binds to basic substances such as alkali proteins in cytoplasm

Question 5

How do cells with increased protein synthesis appear and why?

Answer 5

They appear darker purple

Because they contain more RER and thus are more basophilic

Question 6

Why and how are frozen sections made?

Advantages? Disadvantages?

Answer 6

• made when fast preparation is needed, e.g. intraoperative biopsy
• hardness for sectioning created by freezing, staining can be done immediately

advantages: simple, fast, lipids preserved

disadvantages: sections thicker, 10-20 micrometers, so lower quality

Question 7

How is tissue prep different for electron microscopes?

Answer 7

Basically same, just requires better fixation and thinner sections

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1. **Fixation **- glutaraldehyde used, osmium tetraoxide and/or uranyl sulfate for postfixation (bind to lipids, better contrast)
2. **Dehydration/Clearing - **same, but propylene oxide used for clearing
3. **Embedding **- hard epoxy or acrylic resins
4. **Sectioning **- ultramicrotome, diamond knife, 50-100 nm sections collected on copper grids
5. Removal of Resin
6. **Staining **- heavy metals to increase contrast (lead citrate, uranyl acetate)