Histological fixatives 组织固定剂和固定方法
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Histological fixatives 组织固定剂和固定方法
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).
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
Fixed tissue should be transferred to 70% alcohol.
Carnoy's fluid - fixation time 1-3 hours.
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.
Formalin (~ 40% aqueous solution of formaldehyde) - 5ml
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
Michel's fixative for immunoflourescence - fixation time 24-48 hours.
0.0625g N-ethylmaleimide - HANDLE WITH CARE!
Before use add 55g ammonium sulphate and allow to dissolve.
Adjust pH to 7.0-7.2 with 1M KOH.
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.
Fixed tissue should be washed overnight in running tap water before processing.
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.
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.
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.
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