Staining with Chromoxane Cyanine R

Since Pearse in 1957 introduced chromoxane cyanine R as a dual nuclear and cytoplasmic stain there have appeared numerous procedures for use of this dye. These have differed widely, sharing in common mainly the implication that each is best. A defendable procedure has been developed on an experimental basis and is reported here. Four stock solutions are needs. (1) a 0.2% solution of chromoxane cyanine R in 0.5% aqueous H₂SO₄ (v/v); boil this solution for 5 min, (2) 10% FeCl₃ in 3% HCI, (3) 1% aqueous NH₄OH, and (4) 1% HCI in 70% ethanol. The staining solution: 40 ml of dye solution, 2 ml of FeCl₃ solution, 8 ml H₂O. Dewax and hydrate sections and stain for 10 min. If a myelin sheath stain is desired differentiate for 1 min in solution (3). For a nuclear stain differentiate for 1 min in solution (4). The nuclear stain when counterstained with eosin closely resembles the routine hematoxylin and win. Histochemical tee show that the functional pup for myelin staining contains nitrogen, and probably hydrogen bonding is involved. The nuclear stain involves a different functional group and possibly neither electrostatic nor hydrogen bonding.