[Histonet] Fluorescent dyes and xylene

[John Kiernan]

Most of the Alexa Fluor dyes appear to be trade secrets: only a few are identified with chemical structures in the Molecular Probes/Invitrogen catalogue. Fluorescent labels of known identity do survive dehydration, clearing and mounting in a permanent medium and are still OK years later, stored in boxes at room temperature. It is, of course, necessary to use a non-fluorescent mountant such as DPX or one of the poly(methacrylate) ones (Entellan, Cytoseal etc). 
 
The notion that it's necessary to mount fluorescent preparations in slightly alkaline aqueous media may derive from an influential book by Nairn (1976), which stressed the effects of pH on fluorescence intensity and recommended mounting only in a somewhat alkaline glycerol-water mixture, and storage of slides in flat trays in a fridge.  In fact, fluorescent labels bound to proteins survive dehydration, clearing and permanent mounting (see, eg, Stoddart 1973, Whyte 1978, Allen 1994, Espada 2005, Hertzler 2006). The paper by Espada et al (2005) illustrates this point very well and also discusses reasons why a polystyrene medium (ie DPX) may be best.
 
You would not expect dehydration etc to remove or damage a fluorescently labelled antibody or other protein. Immersing the stained section in alcohol coagulates the protein instantly, along with the covalently attached fluorochrome molecules. 
 
References.
Allen DT, Kiernan JA (1994) Permeation of proteins from the blood into peripheral nerves and ganglia. Neuroscience 99: 755-764. http://www.ncbi.nlm.nih.gov/pubmed/8008217?ordinalpos=33&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum
Espada J, Juarranz A, Galaz S, Canete M, Villanueva A, Pacheco M, Stockert JC (2005) Non-aqueous permanent mounting for immunofluorescence microscopy. Histochem. Cell Biol. 123: 329-334.  http://journals1.scholarsportal.info/details.xqy?uri=/09486143/v123i0003/329_npmfim.xml
Hertzler PL (2006) Rhodamine fluorescence after 15-year storage in methyl salicylate. Microscopy Today 14-2: 48.  http://www.microscopy-today.com/jsp/mto/print_archive/print_archive.faces
Nairn RC (1976) Fluorescent Protein Tracing. 4th ed. Churchill-Livingstone, London. ISBN 0443012733. http://openlibrary.org/b/OL5063508M/Fluorescent-protein-tracing
Stoddart RW, Kiernan JA (1973) Histochemical detection of the alpha-D-arabinopyranoside configuration using fluorescent-labelled concanavalin A. Histochemie 33: 87-94. http://www.springerlink.com/content/vk667214g6m80530/?p=fb8574a99977492fa339fb2300500e29&pi=2
Whyte A, Loke YW, Stoddart RW (1978) Saccharide distribution in human trophoblast demonstrated using fluorescein-labelled lectins. Histochem. J. 10: 417-423. http://www.springerlink.com/content/h710461877mk8010/
 
John Kiernan
Department of Anatomy & Cell Biology
University of Western Ontario
London, Canada.
= = =
----- Original Message -----
From: Bob Nienhuis <bob.nienhuis <@t> gmail.com>
Date: Monday, July 20, 2009 14:10
Subject: [Histonet] Fluorescent dyes and xylene
To: Histonet <histonet <@t> lists.utsouthwestern.edu>

> Will the Alexa Fluor dyes withstand some dehydration and 
> clearing in
> alcohol and xylene?
> 
> If they won't are there any bright fluorescent dyes that can be 
> obtainedcongugated to a secondary antibody that will?
> 
> Looking to immunostain tyrosine hydroxylase in rodent brain with
> a fluorescent marker in tissue that needs to be dehydrated and
> cleared, perhaps with an abbreviated series.
> 
> Bob Nienhuis
> VA / UCLA Neurobiology Research
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