Transcript Research Techniques I (Biology 513)

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Research Techniques I
(Biology 513)
Fixation
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Introduction
 Why
do we fix tissue
 What
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makes an ideal fixative?
Penetrate rapidly and prevent postmortem changes
Coagulate cell contents into insoluble substances
Protect tissue against shrinkage and distortion during
dehydration, embedding and sectioning
Prepare the tissue for staining
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Fixatives
 Realize
there is no ideal fixative
 With
few exceptions most reliable fixatives
are a mixture of:
 A. coagulant
chemicals, and
 B. non-coagulant chemicals
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Bouin fixative
Components
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Formaldehyde – Advantage: fixes cytoplasmic elements,
Disadvantage: retards paraffin penetration
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Picric acid – Advantage: coagulates cytoplasm thus admitting
paraffin, Disadvantage: makes the tissue soft and shrinks the
tissue
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Acetic acid – Advantage: compensates for defects in
formaldehyde and picric acid
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Fixation
Considerations
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1. What is the tissue to be used for?
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2. What is the rate of penetration of the fixative?
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Is a routine all purpose fixative adequate or must some
special part of the cell be preserved?
If the tissue is very dense, then the pieces of tissue must be
as small as possible.
* The ratio of tissue to fixative should be about 1:20
parts per volume.
3. Will the fixative make the tissue too hard?
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If too hard, the tissue may be difficult to section.
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Fixation methods
1.
Perfusion fixation
2.
Immersion fixation
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Immersion fixation
Steps
1.
Remove the tissue from the specimen and place in the
fixative as quickly as possible.
2.
Slice the tissue prior to placing it in the fixative to ensure
optimum fixation in all areas.
3.
If fixing animal tissue, wash off any excess blood as this will
retard the penetration of the fixative.
4.
Never allow the tissue to dry out.
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Immersion fixation
Steps
5.
Leave the tissue at room temperature overnight, this
increases the rate of penetration. Do not leave at room
temperature more than 24 hr.
6.
Leave tissue in fixative for no more than 24hr., then wash
fixative in a buffered solution. Washing prevents the fixative
interfering with subsequent processes.
7.
Washed tissue can be transferred into 70% ethyl alcohol and
stored for several months.
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Post mortem changes