You are on page 1of 11

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.

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.

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.

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.