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Hi Jason,
Sounds like blood, specifically, paraformaldehyde fixed hemoglobin in
RBCs. Are the spots red blood cell size (toy did not specify a scale for
"small"). If much smaller than RBCs, then might be "crystals" of fixed
hemoglobin.
The Billinton and Knight paper cited in Paul Rigby's web site is an
excellent reveiw (Paul's autofluorescence web page had some weird text in
Internet Explorer - looked fine after copying and pasting to Word).
You did not specify how you are imaging the specimens. Consider using an
NIR fluorophore such as QD655, Cy5.5 or Alexa Fluor 647, For nuclear
staining, DRAQ5 or To-Pro-3. I wanted to mention that with our visible light
laser scanning confocal we routinely image 80-150 um into freshly dissected
hemi-sectioned mouse brains. We perfuse in fluorescein-tomato lectin (Vector
Lab) or biotin-tomato lectin + Alexa Fluor 647-streptavidin to label blood
vessels. For best results, perfuse to flush the circulating blood out (i.e.
cardiac puncture, perfuse in the lectin, wait a short time, perfuse with
paraformaldehyde-saline). Brains are imaged with a 10x lens on a Mattek
imaging dish (inverted confocal), kept on ice when not looking, and then
returned to the wet lab for sectioning and conventional tissue staining.
Best wishes,
George
George McNamara, Ph.D.
Imaging Scientist
Congressman Julian Dixon Image Core
The Saban Research Institute of Childrens Hospital Los Angeles
4650 Sunset Blvd., MS 133, SRT 1016
Los Angeles, CA 90027
323-669-2548 voice
323-251-8878 cell
323-668-7921 fax
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-----Original Message-----
From: Jason W [ mailto:[log in to unmask]
<mailto:[log in to unmask]> ]
Sent: Saturday, May 08, 2004 10:31 AM
To: [log in to unmask]
Subject: Autofluorescence after intracerebral hemorrhage
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Hello,
I'm a new member to the list so this is my first message. I am working on a
model of intracrebral hemorrhage in the rat. The hemorrhage is in the
striatum. The rat is perfused with 4% para and then the brain is post fixed
for one night in 4% para and then 1 day in 10% sucrose and then 1 week in
30%
sucrose. The brain is frozen in CO2 and then sliced in a microtome. I have
observed a bright ring of autofluorescence around the hemorrhage 7 days
after
hemorrhage onset. The autofluorescence is not visible in other parts of the
brain. The autofluorescence appears as small, punctate spots. They appear
orange when excited by UV, green when excited by blue and red when excited
by
green. From what I have read they appear to be lipofuscin but I have not
found
any literature discussing the presence of lipofuscin in intracrebral
hemorrhage. Anyone have any experience with this and any suggestions for
quenching it? Thank you very much.
Jason Wasserman
University of Toronto, Dept. of Physiology
Toronto Western Research Institute
Toronto Western Hospital
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