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Hi Paul,

I ordered some 33342, mixed with distilled water, and tried some
freshly excised tissue.  However, I am seeing very poor diffusion into
tissue.  At 1 ug/ml and 2 minutes staining, I literally only stain the
uppermost cell layer in solid tissue (although they stain very well).
I tried much higher concentration and 10 minute soaking, but it made
very little difference.  The diffusion of 33342 into live tissue seems
to be very slow as compared to DAPI.

I suppose what I really want is a cell permanent dye that also has
reasonable diffusion through extracellular space.  Too much to ask for
in the blue?  I could also look at the mid to far red, although my PMT
sensitivity isn't as good there and I'm not imaging deep enough that
reduced scattering will matter very much.

Mike


On Mon, Sep 1, 2014 at 12:28 AM, Paul Rigby <[log in to unmask]> wrote:
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>
> Hi Michael,
> A couple of issues might be contributing to your problems:
> 1. DAPI is usually cell membrane-impermeant. Try using Hoechst 33342 (not Hoechst 33258) - this will get through the cell membrane of live cells.
> 2. Make sure you make your Hoechst/DAPI up as a stock solution in water (definitely NOT phosphate buffer). Also, if you can, use the working solution in a buffer that does not contain phosphate.
> 3. As your cells are live, you might need to extend your incubation time a little more. I would do this in preference to increasing the concentration. You should only need a very short rinse after staining.
>
> Hope this helps.
> Cheers
> Paul
>
> Assoc. Prof. Paul Rigby
> Centre for Microscopy, Characterisation & Analysis (M510)
> The University of Western Australia
> 35 Stirling Highway
> Crawley  WA  6007
> Australia
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Michael
> Sent: Monday, 1 September 2014 6:34 AM
> To: [log in to unmask]
> Subject: Fast DAPI staining protocol
>
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> I'm working on a protocol using DAPI to quickly stain freshly excised tissue.  For now I am testing on formalin fixed tissue, but am seeing a lot of non-specific DAPI staining in some extra-cellular areas of the tissue.
>
> My protocol is basically the recommended dapi protocol from the manufacturer:
>
> 1 mg/ml DAPI stock solution diluted 1000:1 in PBS.
> Stir excised tissue in solution for 60 seconds.
> Stir in rinse solution (also PBS) for 120 seconds.
>
> I would like to keep the staining process as brief as possible, as lengthy staining will be hard with fresh tissue.  Is the problem here that I am using too much DAPI?  Or does the formalin fixation interfere with staining?  Or is DAPI not a great choice for this application?  I know faster agents are available than DAPI for live staining, but I'm counterstaining with some green/red agents as well, so I'm restricted to blue for nuclear staining and haven't seen any good alternatives.