HISTOLOGICAL FIXATIVES
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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)
- Formaldehyde (NP)
- Mercuric chloride (P)
- Methanol (P)
- Osmium tetroxide (NP)
- Picric acid (P)
- Potassium dichromate (NP)
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 (~ 40% aqueous solution of formaldehyde) - 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 (~ 40% aqueous solution of formaldehyde) - 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 (~ 40% aqueous solution of formaldehyde) - 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) - 1000ml
Sodium dihydrogen orthophosphate (monohydrate) - 40g
Disodium hydrogen orthophosphate (anhydrous) - 65g
Distilled water - 9000ml
This fixative is suitable for most histological purposes. It is to be preferred to formol-saline (a single 10% solution of formalin in
0.9% aqueous NaCl) as formalin pigment is avoided. Specimens may be stored in this fluid. The solution is isotonic.
Dissolve the salts in 1000ml of the water before adding to the rest of the constituents.
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 -70o C.
Paraformaldehyde - fixation time depends on technique to follow.
Sodium dihydrogen orthophosphate 2.26% - 41.5ml
Sodium hydroxide 2.52% - 8.5ml
Heat to 60-80o C 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.
Mounting Fluid (for museum specimens)
1. Glycerine 1500ml
2. Sodium acetate 500g
3. Formaldehyde 25ml
4. Distilled water 5000ml
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.
