RE: IF protocols on Ventana

From:Patsy Ruegg

On the DAKO autostainer I put a blanket over the cover on the machine to
keep light out and to keep moisture in.

-----Original Message-----
From: Gayle Callis []
Sent: Tuesday, May 13, 2003 5:54 PM
Subject: IF protocols on Ventana

By IF, you mean immunofluorescent staining?

Photobleaching of fluorophores will be a problem if the system allows slide
exposure to room light during staining procedures. Private communication
with another lady confirmed this with another open to light automatic

Some fluorophores, FITC, Rhodamine are more susceptible to photobleaching
(it is NOT quenching of fluorescence which can be reversed - photobleaching
is the correct term here.) There are different chemical structures for
FITC's/Rhodamines for brighter signal and more resistence to
photobleaching.  The superb Alexa dyes, noted for resistence to
photobleaching (Molecular Probes) must be protected from light during
staining protocols. It may be possible to use the more robust fluorescent
dyes with a very short staining procedure.  Molecular Probes tech services
can answer questions also.

Unless slides can be protected from light exposure during the staining
protocol, then the answer is no unless slides remain totally in the dark
after application of fluorophore.  In our lab for confocal laser scanning
microscopy and fluorescence microscopy, great care is take to protect
diluted antibodies conjugated to fluorescent dyes. These reside in
blackhole drawer after dilution, spun down to remove fluorescent garbage
i.e. protein aggregates conjugated to fluorochrome just before applying to
section, then applied and protected from light to bitter end.

Another possibility is have the machine in a dark room with lights out
during staining or cover it with a big black "tent" blanket - hmmm a tidge
silly!  On confocal listserver, one lady stained in a dark room with
protective light for developing film! That is dedication!

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
S. 19th and Lincoln St
Bozeman MT 59717-3610

406 994-6367 (lab with voice mail)
406 994-4303 (FAX)


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