Obadiah
for Fibrin
The name of this stain comes from the letters OBDR45, which refer to the dyes used: Orange, Blue, Direct Red 45 (a synonym for one of the red dyes).
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. Results are usually poor with formalin fixed material, even if treated with Bouin’s fluid at 56°C for an hour or so.
Materials
- An acid resistant nuclear stain, such as Weigert’s iron hematoxylin, or the celestine blue-hemalum sequence.
- Picro-mercuric ethanol
Material Amount Ethanol, absolute 100 mL Picric acid to saturation Mercuric chloride to saturation - Orange
Material Amount Orange G 0.5 g Phosphotungstic acid 1 g Ethanol, 95% 100 mL - Blue
Material Amount Naphthalene blue black CS 1 g Acetic acid, glacial 1 mL Distilled water 99 mL - Red – Option 1
Material Amount Chicago red 2.5 g Acetic acid, glacial 2.5 mL Distilled water 97.5 mL - Red – Option 2
Material Amount Polar brilliant red BN 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, as this produces tissues that stain poorly. Sections should be 3-5 µ thick.
Protocol
- Dewax sections with xylene.
- Place into a sealed container of trichlorethylene at 56°C for 48 hours.
- Rinse well with absolute ethanol.
- Refix sections in picro-mercuric-ethanol for a minimum of 3 and preferably 24 hours.
- Remove mercury pigment with the iodine-thiosulphate sequence.
- Stain nuclei with an acid resistant nuclear stain.
- Place in the orange stain for 2 minutes.
- Rinse with distilled water.
- Place in the Blue stain up to 30 minutes.
- Differentiate with the polyacid for 5 minutes.
- Place in the Red stain 15-20 minutes (polar brilliant red) or 15-30 minutes (chicago red).
- Rinse with distilled water.
- Dehydrate with ethanol.
- Clear with xylene and mount with a resinous medium.
Expected Results
- Old fibrin – black
- Younger fibrin – may be yellow
- Connective tissue – red
Notes
- Note that this method reverses the usual order and uses a blue stain before the red stain.
- Lendrum considered that this method demonstrated fibrin that other methods stained as collagen.
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.
