[Histonet] Re: Counterstain for fluorescent tissues

[Johnson, Teri]

John, would the neutral red technique work the same with unfixed tissues? The fluorophore Nick uses might not fluoresce after formalin fixation. I suspect he would also need to be sure it's stable after mounting with DPX or other resinous medium.

Teri Johnson, HT(ASCP)QIHC
Managing Director, Histology Facility
Stowers Institute for Medical Research
Kansas City, MO


Neutral red (CI 50040) is an excellent fluorescent Nissl stain: 0.002%, in water, for 5 minutes; dehydrate, clear, and mount in DPX or another non-fluorescent resinous medium. With excitation by either near-UV or blue light (range 325-500nm) the Nissl substance and nuclei fluoresce yellow-orange.

Reference: Allen, D. T. & Kiernan, J. A. 1994. Permeation of proteins from the blood into peripheral nerves and ganglia. Neuroscience 59(3):755-764.
   We used this very dilute neutral red as a fluorescent counterstain for paraffin and cryostat sections of  formaldehyde-fixed tissues from rats that had received iv injections of rhodamine-labelled albumin (green excitation, orange-red emission, localization mostly extracellular).
   Franz Nissl was a man, not a granule, substance or stain, so we should give his surname its capital N. Check out http://www.whonamedit.com.

John Kiernan
Anatomy, UWO
London, Canada
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