[Histonet] Re: Counterstain for fluorescent tissues

[Nicholas David Evans]

Thanks for the help - 

I could just mount in hydromatrix or similar and view immediately without
dehydration. I will try this and will also use DAPI and prepare an H&E on
the adjacent slide to show alongside it. 
Thanks again for the advice,
Nick

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Johnson,
Teri
Sent: Monday, January 11, 2010 10:43 AM
To: 'histonet <@t> lists.utsouthwestern.edu'
Subject: [Histonet] Re: Counterstain for fluorescent tissues

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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