+

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

1. Perfusion fixation

2. Immersion fixation

+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.

+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.

+Post mortem changes