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Ch 8.Immunohistoch / immunology
Ch 10.GC/MS, NMR and Proteomics
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Fixation & Tissue process Protocol (
Histological
Fixatives and Sample Fixation
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) - 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
0.9% 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 -70C.
Paraformaldehyde - fixation time depends
on technique to follow.
Sodium dihydrogen orthophosphate 2.26% - 41.5ml
Sodium hydroxide 2.52% - 8.5ml
Heat to 60-80C 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.
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.