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 (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.
|An acid resistant nuclear stain, such as Weigert's iron hematoxylin,|
|or the celestine blue-hemalum sequence.|
|Naphthalene blue black CS||1||g|
|Acetic acid, glacial||1||mL|
|Chicago red||2.5||g||or||Polar brilliant red BN||1||g|
|Acetic acid, glacial||2.5||mL||Acetic acid, glacial||1||mL|
|Distilled water||97.5||mL||Distilled water||99||mL|
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.
Lendrum, A. C., et. al. (1962)
Studies on the character and staining of fibrin.
Journal of clinical pathology, v. 15, p. 401.
Last updated Oct 2005.