Masson 44/41
for Fibrin

With this trichrome stain full and complete fixation is absolutely essential. Minimalist formalin fixation and quick processing will give poorly stained erythrocytes with red or orange tinges instead of the yellow they should have. Lendrum and coworkers specified about seven days fixation in formal sublimate (9 parts saturated mercuric chloride and 1 part concentrated formalin) followed by processing thoroughly, sectioning, degreasing with trichlorethylene for 48 hours, then refixing in picro-mercuric-ethanol. Results are usually poor with formalin fixed material, even if treated with Bouin's fluid at 56°C for an hour or so.

Solutions

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

or the celestine blue-hemalum sequence.

Picro-mercuric ethanol
Ethanol, absolute	100	mL
Picric acid	to	saturation
Mercuric chloride	to	saturation

Plasma stain
Ponceau 6R	1	g
Acetic acid, glacial	1	mL
Distilled water	99	mL

Fibre stain
Naphthalene blue black CS	1	g
Acetic acid, glacial	1	mL
Distilled water	99	mL

Polyacid
Phosphotungstic acid	1	g
Distilled water	100	mL

Tissue sample
3 mm slices of tissue should be fixed in formol sublimate for one week. They should be paraffin processed with a schedule that thoroughly and completely dehydrates, then thoroughly cleared with xylene and infiltrated with paraffin wax. This process would normally take 48 hours or longer. Err on the side of completeness, and do not attempt to shorten the process. Avoid rapid fixation and overnight processing, as this produces tissues that stain poorly. Sections should be 3-5 µ thick.

Method

1. Dewax sections with xylene.
2. Place into a sealed container of trichlorethylene at 56°C for 48 hours.
3. Rinse well with absolute ethanol.
4. Refix sections in picro-mercuric-ethanol for a minimum of 3 and preferably 24 hours.
5. Remove mercury pigment with the iodine-thiosulphate sequence.
6. Stain nuclei with an acid resistant nuclear stain.
7. Place in the plasma stain for 5 minutes.
8. Differentiate with the polyacid for 5 minutes.
9. Place in the fibre stain for 30 minutes.
10. Rinse briefly with 1% aqueous acetic acid.
11. Dehydrate with ethanol.
12. Clear with xylene and mount with a resinous medium.

Expected results

• Fresh fibrin  –  red
• Older fibrin  –  black
• Connective tissue  –  pale blue
• Plasma cell inclusions  –  red

Notes

1. Ponceau 6R is also known as acid red 44.
2. Naphthalene blue black CS is also known as acid blue 41.

 

Reference
Lendrum, A. C., et. al. (1962)
Studies on the character and staining of fibrin.
Journal of clinical pathology, v. 15, p. 401.