DAPI is not very soluble with phosphate. See the data sheet. They do
mention using it in PBS (at a working dilution, I assume) but don't use
PBS for a stock - big mistake.. trust me....
http://probes.invitrogen.com/media/pis/mp01306.pdf==================================
Preparing the DAPI
Stock Solution To make a 5 mg/mL DAPI stock solution (14.3 mM for the
dihydrochloride or 10.9 mM for the dilactate), dissolve the contents of
one vial (10 mg) in 2 mL of deionized water (dH2O) or dimethylformamide
(DMF). The less water-soluble DAPI dihydro-chloride may take some time
to completely dissolve in water and sonication may be necessary.
Note: Neither of these DAPI derivatives is very soluble in
phosphate-buffered saline (PBS).
====================================
Dale
Mayandi Sivaguru wrote:
>
> Esteban, as you have predicted, it sounds like the old vectashield, have
> you seen any speckles also in the sample fluorescing red, then it might
> be phenylendiamine, the antifade used in vectashield would have
> precipitated (usually a brown precipitate under visible light), but I
> have not seen them under UV light. Using a new batch might answer the
> question.
>
> However, we reccommend, using DAPI always as an aqueous incubation in
> PBS rather than in the mounting medium, for better penetration.
> Shiv
>
> At 04:18 PM 1/20/2009, you wrote:
>> Hi all,
>>
>> One of my users came to me with immuno-stained paraffin sections of
>> mouse cardiac muscle mounted in Vectashield + DAPI (secondary Abs were
>> Alexa488 and Alexa568). The problem is that the nuclei are fluorescing
>> red under UV excitation. Has anyone ever seen this apparent shift in
>> emission of DAPI (e.g. with old Vectashield) or have an idea of what's
>> going on?
>>
>> Thanks,
>> Esteban
>>
>> --
>> G. Esteban Fernandez, Ph.D.
>> Associate Director
>> Molecular Cytology Core Facility
>> University of Missouri
>> 120 Bond Life Sciences Center
>> Columbia, MO 65211
>>
>>
http://www.biotech.missouri.edu/mcc/>>
>> 573-882-4895
>> 573-884-9395 fax
>
> Mayandi Sivaguru, PhD, PhD
> Microscopy Facility Manager
> 8, Institute for Genomic Biology
> University of Illinois at Urbana-Champaign
> 1206 West Gregory Dr.
> Urbana, IL 61801 USA
>
> Office: 217.333.1214
> Fax: 217.244.2496
>
[hidden email]
>
http://core.igb.uiuc.edu>
> <
http://core.igb.uiuc.edu/>
>
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