[Histonet] catecholamine/glyoxylic acid fluoresence

[Young Kwun]

Dear Dr Chris,
Your fluorescence microscope setting seems OK. I used Olympus BH2 equipped
with epi-illumination, and violet excitation and a Y-475 barrier filter.
According to my old thesis, I used 2% glyoxylic acid and 15% sucrose in 0.1M
phosphate buffer, pH adjusted to 5.0 with NaOH for 12 minutes. Magnesium
chloride (0.5%) was added to the solution, immediately before use. After
drying the slides I put into a gassing chamber for 5 min.

Best wishes,

Young








Young Kwun, PhD
Senior Hospital Scientist
Dept of Anatomical Pathology
Concord Hospital
Concord NSW 2139
Australia
Phone:61-2-9767 6075
Fax:61-2-9767 8427
Email:kwuny <@t> email.cs.nsw.gov.au
 

 

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Chris
Johnson
Sent: Tuesday, 23 November 2004 11:18 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] catecholamine/glyoxylic acid fluoresence

Dear All,
 
I have recently been trying to show up sympathetic innervation of blood
vessels (noradrenaline/norepinephrine. I have used rat tail artery
(i.e., good sympathetic innervation) incubated in phosphate buffer and 2
% glyoxylic acid pH’d to 7.4 for 90 minutes followed 4 minutes at 100
ºC. At best I have seen very faint fluorescence with the very slightest
hint of a nerve plexus. I don’t think it is the processing as I have
used it successfully before and it seems to be fairly robust. I suspect
it is our filtering, but on looking at the literature, I’m not sure.  I
am using 
Excitation          380 – 400 nm
Dichromatic       440 nm high pass
Emission           455 – 485 nm
 
What are the emission characteristics of the fluorophore from this
reaction?
What am I doing wrong?!!!!
Any suggestions would be greatly appreciated!!
 
Best wishes,
 
Chris
 
 
Dr Christopher Johnson
Department of Physiology,
Medical Biology Centre, 
Queen's University of Belfast,
97 Lisburn Rd
Belfast BT7 9BL
02890 272092
 
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