>Date: Thu, 19 Aug 1999 14:36:42 -0700
>From: Rose_Bellantoni@integra-ls.com
>Subject: Catacholamine fluorescence
>To: histonet@pathology.swmed.edu
>Mime-Version: 1.0
>
>Histonetters,
>
>I am posting this question for a friend of mine.  He is looking for a
>method for fluorescent detection of catecholamines in brain tissue.  He has
>a reference for a Falck-Hillarp method that was publised around 1962.  It
>involves formaldehyde and glyoxilic acid, but  the original method is
>referenced without the description of the procedure.  He would greatly
>appreciate your help.  And I would get a free lunch to boot!
>
>Lots of thanks.
>
>Rose Bellantoni
>
>Integra LifeSciences, CRC
>tel:  619-622-2740
>fax: 619-535-8269

Rose,
	Enjoy your lunch. Any problems let me know.
Ian.
>
NORADRENALINE (Formaldehyde-induced Fluorescence).
Eranko,O. (1955) Nature, Lond. 175, 88.
Falck,B. & Owman,C. (1965) Acta Univ. Lund., Sect., II, No.7
Pearse,A.G.E. (1972) HISTOCHEM. Theor. & Appl. 3rd.ed. Vol.2 Ch.27.

METHOD.
  1.) Quench small pieces of tissue (1-4 mm) in Iso-pentane cooled with
liquid nitrogen.
  2.) Transfer to a freeze dryer or store the frozen tissue in a liquid
nitrogen fridge .
  3.) Freeze dry tissue overnight at -60oC.
  4.) Transfer quickly to a flat bottomed powder bottle with a layer of
paraformaldehyde at the bottom.
	Use fresh paraformaldehyde for each freeze dry.
  5.) Vapour fix for 1-3 hours at 60oC.
  6.) Transfer quickly to molten paraffin wax and infiltrate for 1-2 hours.
  7.) Embed in fresh wax.
  8.) Cut sections and mount on clean slides.
  9.) Transfer to a 60oC hotplate to melt wax and allow sections to spread.
10.) Allow wax to harden again.

Either;
11.) Mount using liquid paraffin.
12.) Warm on a hotplate for 20-30 minutes to dissolve wax in the liquid
paraffin.
13.) Examine using UV with a 480nm filter.

Or;
11.) Mount with a mixture of 3 ml. xylene and 30 ml. Entellan (Merck).
12.) Warm on a hotplate for 20-30 minutes to dissolve wax in the mountant.
13.) Examine using UV with a 480nm filter.

RESULT.
	Blue-green fluorescence in adrenergic endings (NA-FIF).

Dr. Ian Montgomery,
West Medical Building,
University of Glasgow,
Glasgow,
G12 8QQ,
Scotland.
Tel: 0141 339 8855 Extn. 6602.
Fax: 0141 330 4100.
e-mail: ian.montgomery@bio.gla.ac.uk