Slidders’ Fuchsin-Miller
for Fibrin

Slidders’ fuchsin-miller is a technique belonging to a group that Lendrum and his co-workers referred to as "Yellowsolve" methods, because they use a yellow dye dissolved in cellosolve (2-ethoxy-ethanol), a less polar solvent than water, to differentiate the plasma stain and stain connective tissue. In this respect they are similar to the HPS (which uses ethanol) and the phloxine-tartrazine method (which also uses cellosolve). In yellowsolve methods the red stained fibrin is differentiated with a polyacid, then any dye remaining in other tissue components is displaced by the yellow dye. The low polarity of the solvent causes this to proceed more slowly, allowing some control.

As with most of the techniques for fibrin which depend on displacement, overnight mercuric chloride fixation (formol sublimate, B5) is preferred, followed by overnight paraffin processing. Overnight formalin fixation and paraffin procesing can produce acceptable results if sections are refixed for an hour in Bouin’s picro-acetic-formalin mixture at 56°C. Optimal results are obtained with extended mercuric chloride fixation, thorough processing, degreasing and secondary fixation of sections, as for the Picro-Mallory.

Solutions

An acid resistant nuclear stain, such as Weigert's iron hematoxylin,

or the celestine blue-hemalum sequence.

Fuchsin
Acid fuchsin	1	g
Acetic acid, glacial	2.5	mL
Distilled water	100	mL

Miller
Milling yellow 3G	2.5	g
2-ethoxy-ethanol	100	mL

Phosphotungstic acid
Phosphotungstic acid	1	g
Distilled water	100	mL

Tissue sample
3 mm slices of tissue should be fixed in formol sublimate (or B5) overnight. Paraffin process overnight. Overnight formalin fixation is usually satisfactory, but avoid rapid fixation with formalin and short processing, as this produces tissues that stain poorly even with the secondary fixation specified. Sections should be 3-5 µ thick.

Method

1. Bring sections to water via xylene and ethanol.
2. If mercury fixed, remove with the iodine-thiosulphate sequence.
3. If not mercury fixed, place into Bouin's fluid at 56°C for 1 hour.
4. Rinse well with distilled water.
5. Stain nuclei with an acid resistant nuclear stain.
6. Rinse with 95% ethanol.
7. Place in fuchsin for 10 minutes.
8. Rinse with distilled water.
9. Differentiate with phosphotungstic acid for 5 minutes.
10. Rinse well with distilled water.
11. Blot.
12. Rinse well with cellosolve.
13. Place into milling yellow until fibrin is red and tissues are yellow.
    This step takes from ½ - 4 hours.
14. Rinse briefly with 1% aqueous acetic acid.
15. Rinse with cellosolve.
16. Clear with xylene and mount with a resinous medium.

Expected results

• Fibrin  –  red
• Other tissue  –  yellow
• Nuclei  –  black

Notes

1. 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.
2. 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.
3. 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.