Transcript Research Techniques I (Biology 513)

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Research Techniques I
(Biology 513)
Tissue processing
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Step 1: Dehydration
 Why
dehydrate tissue
Answer: hydrated tissue is soft and contains
hollow spaces (lumen) which deform upon
sectioning
 To
prevent deformation, hollow areas are
replaced by a medium (either wax or
plastic)
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Step 1: Dehydration
Procedure : process tissue through a series of
graded alcohols. (i.e., 30, 50, 80%)
Problem – dehydration causes tissue
shrinkage
 To
minimize shrinkage process tissue
through more steps of graded alcohols
(i.e., 30, 50, 70, 80, 95%)
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Step 1: Dehydration
Two factors to consider when dehydrating
(i) Time in each alcohol step should be no
more than 1 hr. Longer times leads to tissue
brittleness
(ii) Size counts. The smaller the tissue
section, the less time needed in each
alcohol step
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Step 2: Clearing
Why clear the tissue?
Answer: alcohol will not mix or dissolve with
molten paraffin. Tissue is immersed in
some fluid that is miscible with both alcohol
and paraffin.
Common clearing agents
xylene, toluene, you will use histoclear
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Step 2: Clearing
Problems
Clearing agents harden tissue, hence times
must be minimal (up to 1 hr)
Rapid evaporation, hence stopper bottles
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Step 3: Paraffin infiltration
 Different
types of waxes melt at different
temperatures. Range is 50 – 68 °C
 For
5 µm sections use a wax with a melting
point of 56 – 58 °C
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Step 3: Paraffin infiltration
Problems
 Temperature
regulation is critical.
Overheating paraffin will destroy some of
its properties and reduce sectioning quality
 Limit
the time tissue stays in contact with
hot paraffin (up to 1 hr). Heat causes tissue
shrinkage
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Step 3: Paraffin infiltration
 To
aid in paraffin penetration, a warm
vacuum oven may be used. (i.e., air
trapped in lung tissue is removed in this
manner)
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Step 4: Embedding
Which embedding medium to use?
Paraffin wax

Advantage: can section tissue 2 – 15 µm thick
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Disadvantage: heat required, tissue shrinkage (25%)
Plastics
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Advantage: no heat required, minimal shrinkage
(< 10%)

Disadvantage: narrower range of section thickness than
paraffin (2 – 10 µm)
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Step 4: Embedding
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Step 4: Embedding
(iv) A second disposable (plastic) mold must be placed above
the original mold, to enable the tissue to be sectioned.
** wax for this second mold must be cured at the same time
as the original mold **
(v) Paraffin wax contains 7 – 15% dissolved air. Cooling too
slowly or rapidly will lead to uneven pocketing of air
molecules, resulting in difficulty in sectioning
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Step 5: Sectioning
1. Trim the block into squares or rectangles to reduced the
amount of wax around the tissue
2. Make certain the microtome is set to cut 5 µm sections
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Step 5: Sectioning
3.
When cutting the tissue orient the tissue as shown below
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Step 5: Sectioning
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Step 5: Sectioning
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Step 6: Mounting sections
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The placing of sections onto glass slides
Procedure
1. Clean slides with 95% ethyl alcohol
2. BEFORE placing sections onto slides label the slide on the
frosted side. Use a pencil only (will not wash off later)
3. After cutting the sections, use a heated (45 °C) water bath
(the technique will be demonstrated) to place the sections
onto the slides and allow them to dry overnight. Keep free
from dust
4. Heat fix sections onto slides (65 °C for 30 min.). Allow to
cool