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Research Techniques I (Biology 513). Fixation. Introduction. Why do we fix tissue What makes an ideal fixative? Penetrate rapidly and prevent postmortem changes Coagulate cell contents into insoluble substances
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Research Techniques I (Biology 513) Fixation
Introduction • Why do we fix tissue • What 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
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
Bouin fixative Components • Formaldehyde – Advantage: fixes cytoplasmic elements, Disadvantage: retards paraffin penetration • Picric acid – Advantage: coagulates cytoplasm thus admitting paraffin, Disadvantage: makes the tissue soft and shrinks the tissue • Acetic acid – Advantage: compensates for defects in formaldehyde and picric acid
Fixation Considerations • 1. What is the tissue to be used for? • Is a routine all purpose fixative adequate or must some special part of the cell be preserved? • 2. What is the rate of penetration of the fixative? • 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? • If too hard, the tissue may be difficult to section.
Fixation methods • Perfusion fixation • Immersion fixation
Immersion fixation Steps • Remove the tissue from the specimen and place in the fixative as quickly as possible. • Slice the tissue prior to placing it in the fixative to ensure optimum fixation in all areas. • If fixing animal tissue, wash off any excess blood as this will retard the penetration of the fixative. • Never allow the tissue to dry out.
Immersion fixation Steps • Leave the tissue at room temperature overnight, this increases the rate of penetration. Do not leave at room temperature more than 24 hr. • Leave tissue in fixative for no more than 24hr., then wash fixative in a buffered solution. Washing prevents the fixative interfering with subsequent processes. • Washed tissue can be transferred into 70% ethyl alcohol and stored for several months.
