Picro-Mallory
for Fibrin – Long Version
Lendrum, Fraser and others published several fibrin stains, the most well known being the Picro-Mallory. There are several variants of the method, some more complicated than others.
See this alternative protocol for a shorter version.
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 followed by processing thoroughly, sectioning, degreasing with trichlorethylene for 48 hours, then refixing in picro-mercuric-alcohol. Adequate results can be obtained with thorough formalin fixation (24 hours), thorough processing (overnight), sectioning, followed by refixing in Bouin’s fluid at 56°C for an hour or so. However, results are inferior to those obtained by the full procedure. It is also a method where experience is required for optimal results.
Materials
- An acid resistant nuclear stain, such as Weigert’s iron hematoxylin, or the
celestine blue-hemalum sequence. - Yellow mordant
Material Amount Picric acid, sat. in 80% ethanol 100 mL Orange G 0.2 g Lissamine yellow 0.2 g - Stock differentiator
Material Amount Picric acid 2.5 g Ethanol, 95% 100 mL Phosphotungstic acid 25 g Dissolve ingredients and filter.
- Red differentiator
Material Amount Stock differentiator 40 mL Ethanol, 95% 20 mL Distilled water 90 mL - Blue differentiator
Material Amount Stock differentiator 10 mL Distilled water 90 mL - Red stain
Material Option 1 Option 2 Option 3 Acid fuchsin 1 g – 0.4 g Lissamine fast red – – 0.2 g Biebrich scarlet – 1 g – Acetic acid, glacial 1 mL 1 mL 1 mL Distilled water 99 mL 99 mL 99 mL - Blue stain
Material Amount Methyl blue 1 g Acetic acid, glacial 1 mL Distilled water 99 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, especially with formalin fixatives, as this produces tissues that stain poorly even with secondary fixation such as Bouin’s fluid at 56°C for an hour. Sections should be 3-5 µ thick.
Protocol
- Bring sections to water via xylene and ethanol.
- Remove mercury pigment with the iodine-thiosulphate sequence.
- Stain nuclei with an acid resistant nuclear stain.
- Place in yellow mordant for 2-3 minutes.
- Wash in distilled water until only erythrocytes are yellow.
- Place in the red stain for 5-10 minutes.
- Rinse with 1% aqueous acetic acid.
- Differentiate with the red differentiator until fibrin is prominent microscopically.
- Rinse well in distilled water.
- Place in the blue stain for 5 minutes.
- Rinse briefly with 1% aqueous acetic acid.
- Place in the blue differentiator for 1-2 minutes.
- Biefly rinse with 1% aqueous acetic acid.
- Dehydrate with ethanol.
- Clear with xylene and mount with a resinous medium.
Expected Results
- Fibrin – clear red
- Erythrocytes – yellow
- Connective tissue – blue
Notes
- The most common dye combination is acid fuchsin and methyl blue.
- Adequate results may be obtained with 24-48 hours fixation in neutral buffered formalin, overnight paraffin processing and secondary fixation of sections with Bouin’s picro-formol-acetic for 1 hour at 56°C.
- Good results are obtained with the fixation and processing recommended in the text.
- Optimal results are obtained with the fixation and processing recommended in the text, followed by degreasing and secondary fixation in picro-mercuric ethanol.
Replace step 1 with the following:
- Dewax sections with xylene.
- Place in trichlorethylene in a sealed container at 56°C for 48 hours.
- Rinse sections well with absolute ethanol.
- Place in picro-mercuric-ethanol for 24 hours.
- Wash sections with water, and continue from step 2.
Safety Note
Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.
References
- Lendrum, A. C., et. al. (1962)
Studies on the character and staining of fibrin.
Journal of clinical pathology, v. 15, p. 401.
