-----Original Message-----
From: Cynthia Favara [mailto:cfavara@niaid.nih.gov]
Sent: 04 December 2001 15:05
To: 'Abigail.Mackey'; 'histonet@pathology.swmed.edu'
Subject: RE: fluorescent in situ zymography

Abigail,

No doubt you will get many responses but here are my thoughts.

Non specific fluorescence is coming from the gelatin? Have you tried the procedure on a slide with no tissue just the OCT or whatever you are using to cut your frozens, some interaction there could be a problem.

Sounds interesting.

Cynthia Favara

-----Original Message-----
From: Abigail.Mackey [mailto:Abigail.Mackey@ul.ie]
Sent: Tuesday, December 04, 2001 4:22 AM
To: 'histonet@pathology.swmed.edu'
Subject: fluorescent in situ zymography

Dear all,
I'm working on a method using a Molecular Probes fluorescent marker (oregon
green) to detect gelatinase enzyme activity in frozen muscle tissue
sections. Briefly, gelatin which has a fluorescent marker attached is added
to the section and where gelatinase in the tissue binds to the gelatin, the
gelatin-oregon green is cleaved and becomes fluorescent.
The problem I have is that there are  fluorescent "speckles" visible all
over the section and on the slide surrounding it. These specks do not appear
to be emebedded in the section like the actual signal but rather "sitting"
on top. They can be focussed on when the section itself is out of focus and
vice versa. My negative control (no gelatin but otherwise treated as sample)
does not exhibit this interference. I have tried filtering all my solutions
but this makes no difference.

briefly the method is as follows:
allow sections to thaw and dry at room temp
incubate with gelatin in buffer for 3 hrs at 37C
wash with 1% triton x-100
rinse with distilled water
mount and coverslip

any help at this stage would be appreciated!
many thanks,
abigail

_________________________________
Abigail Mackey
Department Sport and Exercise Sciences
University of Limerick
Ireland

tel +353 (0)61 20 28 84
fax +353 (0)61 33 04 31