We use To-Pro-3 as a counterstain in cytogenetic preps when we use FITC and
Texas Red labelled probes in dual labelled FISH.  We use the stain at a
concentration of 1/1000 diluted in vectorshield and it is made up fresh each
time.  There is substantial bleed through to the red channel so we capture
all the images sequentially and then merge (we use a 1024 as well). It makes
a good block stain but very little resolution is seen. (If the bleed through
is strong you can usually visualise the cells on the microscope - but we
also add DAPI which can be seen with appropriate filters down the microscope
but not picked up by the lasers). What about using a Cy-5 conjugated
antibody for one of your markers and PI as the nuclear stain.

Best wishes

Elaine Levy




At 11:38 28/01/97 +0100, you wrote:
>I'm interested in using the ultra-red dyes TOTO3 and TO-PRO3 (from Molecular
>Probes) for the nuclear staining of cells spun on slides. My goal is to
>simultaneously visualize rsGFP-PML (488/522), another nuclear antigen
>stained by a Texas-red-conjugated Ab (568/605), and nuclear morphology
>(TOTO/TO-PRO staining, 647/670) with a 3-PMT Bio-Rad 1024 confocal apparatus.
>Does anyone have suggestion/experience on TOTO3 and TOPRO3 dyes for nuclear
>staining (fixation, dilution, time of staining..)?
>Thanks in advance,
>
>
>
>Ildo Nicoletti, MD         E-mail: [log in to unmask]
>  I.M.I.S.O. Perugia University Medical School
>   Policlinico Monteluce, 06122 Perugia,Italy
>Phone +039-75-5783974         Fax +039+75+5783444
>           www2.med.unipg.it/imagelab
>
--------------------------------------------------------------
Elaine Levy PhD
Cytogenetics Group
Wellcome Trust Centre For Human Genetics
Windmill Rd.,
Oxford OX3 7BN

tel 01865 740022
fax 01865 742186
email [log in to unmask]
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