Tissue sample
3 mm slices of tissue should be fixed in formol sublimate (or B5) overnight. Paraffin process overnight. Overnight formalin fixation and paraffin processing can produce acceptable results if sections are refixed for an hour in Bouin’s picro-acetic-formalin mixture at 56°C. Avoid rapid fixation with formalin and short processing, as this produces tissues that stain poorly even with the secondary fixation specified. Optimal results are obtained with extended mercuric chloride fixation, thorough processing, degreasing and secondary fixation of sections. Sections should be 3-5 µ thick.
If not mercury fixed, place into Bouin's fluid at 56°C for 1 hour.
Rinse well with distilled water.
Stain nuclei with an acid resistant nuclear stain.
Rinse with 95% ethanol.
Place in fuchsin for 10 minutes.
Rinse with distilled water.
Differentiate with phosphotungstic acid for 5 minutes.
Rinse well with distilled water.
Blot.
Rinse well with cellosolve.
Place into milling yellow until fibrin is red and tissues are yellow. This step takes from ½ - 4 hours.
Rinse briefly with 1% aqueous acetic acid.
Rinse with cellosolve.
Clear with xylene and mount with a resinous medium.
Expected results
Fibrin – red
Other tissue – yellow
Nuclei – black
Notes
It is very important that the milling yellow solution be completely anhydrous. Even small amounts
of water or other solvents can cause problems with displacement and incomplete removal of red dye.
For that reason, step 12 should be thorough and the milling yellow should be used in a Coplin jar with a lid
sealed with tape.
A well stained section would show red fibrin only, muscle and erythrocytes being yellow.
With poorly fixed material, both erythrocytes and muscle fibres may resist displacement of the red dye and
they may have some residual red colouring. Sometimes this may be overcome by prestaining either with saturated picric
acid in absolute ethanol or the MSB martius yellow solution for two minutes immediately
before step 7, but a better resolution is correct fixation and processing.
Some intracellular materials may stain red, such as Paneth cell granules.
Reference
Drury, R.A.B. and Wallington, E.A., (1980) Carleton's histological technique Ed. 5
Oxford University Press, Oxford, UK.
Bancroft, J.D. and Stevens A. (1982) Theory and practice of histological techniques Ed. 2
Churchill Livingstone, Edinburgh & London, UK.
