Thanks for all the feedback from the
listserv. I have been using Vectashield+DAPI, and my next step was to use DAPI
in the secondary AB procedures. We have beautiful labeling with other nuclear
markers in other channels. I have checked the alignment, but I’m writing
a How-To for the protocol binder before I leave, so I’ll do it again today.
Thanks for all the replies,
-
From: Confocal
Microscopy List [mailto:[hidden email]] On Behalf Of Keith Morris
Sent: Wednesday, June 24, 2009
4:18 AM
To:
[hidden email]
Subject: Re: Nuclear Dyes at
405nm?
Hi
Granted the DAPI
excitation is at the tail of the excitation peak*** when using the 405nm laser
line, but there’s a heck of a lot of labeled DNA in the nucleus to
excite. The 405nm laser is essentially designed as a cheap replacement for an
incredibly expensive water cooled uV laser [£50k+ including cooling system] and
this 405nm line is still rarely used for anything other DAPI imaging. Hoechst is actually even worse than DAPI with the 405nm laser
line, down from 11% to nearer 5% on the excitation peak tail.
In every case I have
encountered where the 405nm line laser is consistently very poor at imaging it
requires a long visit by the Zeiss or Leica engineer [for our German confocal
systems]*. Fortunately our confocal systems are on maintenance contracts as
generally an awful lot of DAPI associated hardware seems to been replaced
during ‘repair’ [e.g. lasers, AOTF & other optics]. This
assumes you have done the most a user can do, like align pinholes [for Zeiss
510s], etc… I always have a perfectly DAPI/FITC/TRITC labeled Invitrogen
BPAE cell and kidney slice** to hand to either demonstrate to users that
‘the system is working perfectly and it’s their poor samples’
or, more rarely, that ‘we have a problem Huston’. The BPAE cells
preps seem to store better than the kidney slice, where in the latter the
fluorescence diffuses to ‘incorrect’ locations over time [it stays
bright though]. These Invitrogen slides cost approx £80 and £130 respectively
and last up to a year stored in the fridge/freezer.
As other threads
have pointed out, DAPI labeling isn’t always perfectly straight-forward.
I’d advise staining separately for DAPI and avoid the Likes of
‘Vectashield+DAPI’ to keep the background to the absolute minimum
for top notch DAPI images- although use of the latter shouldn’t produce
really naff ‘off’ DAPI images
Keith
*Assuming it was
decent at installation – and with our Zeiss 510 and Leica SP2 confocals
DAPI [and even Hoechst] imaging is excellent. We image at around 10% laser
power [30mW 405nm laser].
**http://www.well.ox.ac.uk/cytogenetics/Zeiss.shtml
see our Invitrogen slide images
***http://www.invitrogen.com/site/us/en/home/support/Research-Tools/Fluorescence-SpectraViewer.reg.uk.html
---------------------------------------------------------------------------
Dr Keith J. Morris,
Molecular Cytogenetics and Microscopy Core,
Laboratory 00/069 and 00/070,
The Wellcome Trust Centre for Human Genetics,
Telephone: +44 (0)1865 287568
Email: [hidden email]
Web-pages: http://www.well.ox.ac.uk/cytogenetics/
From: Confocal
Microscopy List [mailto:[hidden email]] On Behalf Of Perry, Griffin M
Sent: 23 June 2009 21:31
To:
[hidden email]
Subject: Nuclear Dyes at 405nm?
Dear Listserv,
I was wondering if anyone had better results with DAPI @
405nm than I have. It seems…off. I know it is only getting
excited @ 25%, but I know another group that gets good images at the same
wavelength. The staining has been uneven with DAPI, but with Topro-3 in
the 638nm range, the staining has been beautiful with clearly defined
nucleoli. Does anybody use Hoechst 34580? Are there any
other alternatives to DAPI @ the 405nm range?
Thanks in advance,
-
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