Fixation:

Confers chemical stability on the tissue
Hardens the tissue (helps further handling)
Halts enzyme autolysis
Halts bacterial putrefaction
May enhance later staining techniques
Introduces a 'consistent artefact'

Classification:

Fixatives may be classed as precipitant (P) or non-precipitant (NP) according to

their effect on tissue protein.
Primary fixatives are:
Acetic acid (NP)
Chromium trioxide (P)
Ethanol (P)
Formalin (NP)
Mercuric chloride (P)
Methanol (P)
Osmium tetroxide (NP)
Picric acid (P)
Potassium dichromate (NP)

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Acetic alcohol - fixation time 1 minute.

(For smears, cytospin preparations or frozen sections).

95% methanol - 100ml

Glacial acetic acid - 3ml.

The sections should be washed in water before staining.

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Bouin's fluid - fixation time 6 hours.

Saturated aqueous solution of picric acid - 75ml

Formalin - 25ml

Glacial acetic acid - 5ml

Fixed tissue should be transferred to 70% alcohol.

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Carnoy's fluid - fixation time 1-3 hours.

Ethanol - 60ml

Chloroform - 30ml

Glacial acetic acid - 10ml

Fixed tissue should be processed immediately or transferred to 80% alcohol.

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Formol sublimate - fixation time 4-6 hours.

Formalin - 100ml

Mercuric chloride (saturated aqueous) - 900ml

Fixed tissue should be transferred to 80% alcohol.

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Helly's fluid - fixation time 12-24 hours.

Stock solution:-

Potassium dichromate - 25g

Mercuric chloride - 50g

Sodium sulphate - 10g

Distilled water - 1000ml

For use:-

Stock solution - 100ml

Formalin - 5ml

The fixative solution should be made up just before use. Fixed tissue must be

washed for 24 hours in running tap water prior to
processing.

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Neutral buffered formalin - fixation time 12-24 hours.

Formalin (40% aqueous solution of formaldehyde) - 100ml

Sodium dihydrogen orthophosphate (monohydrate) - 4g

Disodium hydrogen orthophosphate (anhydrous) - 6.5g

Distilled water - 900ml

This fixative is suitable for most histological purposes. It is to be preferred to

formol-saline (a single 10% solution of formalin in
9% aqueous NaCl) as formalin pigment is avoided. Specimens may be stored in this

fluid. The solution is isotonic.

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Michel's fixative for immunoflourescence - fixation time 24-48 hours.

Buffer :

0.81g potassium citrate

0.0625g N-ethylmaleimide - HANDLE WITH CARE!

0.123g magnesium sulphate

100mls distilled water

Before use add 55g ammonium sulphate and allow to dissolve.

Adjust pH to 7.0-7.2 with 1M KOH.

Place tissue biopsies in fixative for 24-48 hours.  Wash tissues in buffer, three

times over 10 minutes, and freeze at -70oC.

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Paraformaldehyde - fixation time depends on technique to follow.

Sodium dihydrogen orthophosphate 2.26% - 41.5ml

Sodium hydroxide 2.52% - 8.5ml

Heat to 60-80oC in a covered container.

Add 2g `Analar' paraformaldehyde and stir until dissolved. Filter.

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Zenker's fluid - fixation time 4-24 hours.

Distilled water - 950ml

Potassium dichromate - 25g

Mercuric chloride - 50g

Glacial acetic acid - 50g

Fixed tissue should be washed overnight in running tap water before processing.

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REMOVAL OF FIXATION PIGMENTS

Formalin pigment

1. Dewax the sections, rinse in 100% alcohol, rinse in 70% alcohol, rinse in

distilled water.

2. Treat in saturated alcoholic picric acid for 30 minutes to 2 hours.

3. Wash well in running tap water.

4. If yellow staining of the section persists rinse in dilute lithium carbonate.

5. Rinse in tap water.

6. Continue with method.

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Mercury pigment

1. Dewax the sections, rinse in 100% alcohol, rinse in 70% alcohol, rinse in

distilled water.

2. Treat in Lugol's iodine for 2 minutes.

3. Decolourise in 5% sodium thiosulphate for 5 minutes.

4. Wash well in running tap water.

5. Continue with method.

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Dichromate pigment

1. Dewax the sections, rinse in 100% alcohol, rinse in 70% alcohol, rinse in

distilled water.

2. Treat in 2% HCl in 70% alcohol 16-24 hours.

3. Rinse in tap water.

4. Continue with method.