BIOL123 Lecture Notes - Lecture 10: Osmium Tetroxide, Fume Hood, Xylene
17 Jun 2018
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• Fixation: maintenance of tissue architecture by cross linking proteins and
inhibiting autolysis
• Procedure:
1. Large specimen is received
2. Specimen is cut into smaller pieces
3. L/M: dipped in formalin or E/M dipped in glutaraldehyde and
then osmium tetroxide (both take 24 hours)
• Dehydration and clearing: excludes water from sample because paraffin
(embedding medium) is immiscible in water
• Dehydration: solution is placed in increasing concentrations of
alcohol beginning with 50% to 100%. Each step takes ~2-3 hours
• Clearing: involves removing the alcohol and replacing it with a
chemical that is miscible in both alcohol and paraffin
• Xylene solution is used
• Carcinogenic solvent
• Use in fume hood
• Smaller tissues ~1 hour
• Large tissues ~2-4 hours
Embedding: tissues are immersed in heated liquid paraffin that permeates
the material
• Larger tissue samples being embedded take longer for the paraffin to
completely permeate the specimen
• If incomplete embedding occurs, sectioning will be effected
• Temperatures of ~52-60 degrees Celsius used to evaporate Xylene
• Block of tissue embedded in paraffin is obtained
• Light microscope: paraffin and plastic resin
• Electron microscope: resin as embedding medium
• Advantages of paraffin: stains reliability & easy to handle
• Disadvantages of paraffin: thin slices are hard to cut without
shattering
•
Sectioning:
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