Supravital staining of murine brain with methylene blue according to the Cajal method: a simple and reliable preparation technique for embedding in paraffin.

Saturated methylene blue solutions or powder were applied with a brush to fresh, unfixed brain slabs. The specimens were then exposed to ambient oxygen in a moist chamber. Fixation was done by application of an ammonium heptamolybdate solution followed by a phosphate-buffered paraformaldehyde/glutaraldehyde mixture with phosphomolybdic acid added. The specimens were dehydrated in tert-butanol before embedding in paraffin. Because of its high specificity for perikarya and for varicose, unmyelinated nerve fibres, this technique is a helpful supplement to other histological methods, particularly to myelin staining techniques. It is therefore well-suited for routine use in neuroanatomical research.