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Histological fixatives. University of Bristol, Department of Clinical Veterinary Science.
 
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HISTOLOGICAL FIXATIVES

Fixation:

  1. Confers chemical stability on the tissue
  2. Hardens the tissue (helps further handling)
  3. Halts enzyme autolysis
  4. Halts bacterial putrefaction
  5. May enhance later staining techniques
  6. Introduces a 'consistent artefact'


Classification:


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.


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.


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.


Formol sublimate - fixation time 4-6 hours.

Formalin - 100ml

Mercuric chloride (saturated aqueous) - 900ml

Fixed tissue should be transferred to 80% alcohol.


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.


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.


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.


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.


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.


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.


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.


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.



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