Fast DAPI staining protocol

[Michael Giacomelli]

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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.

[George McNamara]

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Hi Michael,

If you are lucky, ou will get beautiful DAPI:Phosphate crystals on step
1. If you don't see crystals, try 10 mg/mL out of the bottle. Even if
you don't use it in your experiments (ex. dilute into distilled water),
take some images and send them to the Nikon, Olympus, NSF etc
microscopy/imaging contests. Please acknowledge the listserv when you
win and post the winning web page to here.

You might be better off with 10x higher concentration of DAPI - maybe
better Hoecht 33258 or 33342 - "the law of mass action always wins".

best wishes,

George


On 8/31/2014 5:34 PM, Michael wrote:

> *****
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> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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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.
>
>    

--



George McNamara, Ph.D.
Single Cells Analyst
L.J.N. Cooper Lab
University of Texas M.D. Anderson Cancer Center
Houston, TX 77054
Tattletales http://works.bepress.com/gmcnamara/42

[Paul Rigby-2]

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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:[hidden email]] On Behalf Of Michael
Sent: Monday, 1 September 2014 6:34 AM
To: [hidden email]
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.  

[Stanislav Vitha-2]

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If you have access to a lab microwave with continuously adjustable power
(we use the Pelco Biowave from Ted Pella), you should be able to get
reasonable nuclear staining in live or unfixed tissues.
I never tried this for unfixed tissue, but something like 6 minutes at 150W
should work, especially when combined with a vacuum cycle (vacuum
on/off every 30 seconds)
See figure 6 here:
http://www.tedpella.com/microwave_html/Immunolabeling-Protocol.htm
 It actually lists the non-permeable Hoechst 33258 dye; my guess is that
DAPI would work about the same.
 
No commercial interest, just a satisfied user.

Stan


Dr. Stanislav Vitha      
Microscopy and Imaging Center
Texas A&M University


On Mon, 1 Sep 2014 12:28:55 +0800, Paul Rigby
<[hidden email]> wrote:

>*****
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>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>Post images on http://www.imgur.com and include the link in your
posting.
>*****
>
>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:[hidden email]] On Behalf Of Michael
>Sent: Monday, 1 September 2014 6:34 AM
>To: [hidden email]
>Subject: Fast DAPI staining protocol
>
>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>Post images on http://www.imgur.com and include the link in your
posting.
>*****
>
>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.  

[Vitaly Boyko]

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Dear List,

Would someone advice on the best way to mount a heavy iXon 897 Ultra camera together with a filter wheel to a bottom port a scope. iXon has 8 mounting points, 4 on each side. It could be too heavy for using C-mount only

Thank you,

Vitaly

[WAINWRIGHT James]

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[Manufacturer Response]

Hi Vitaly,

I work for Andor as a Microscopy Support Engineer and have seen at least two instances of our cameras on bottom ports - one on a Zeiss Axiovert (iXon EMCCD) and one on an Olympus IX (Neo sCMOS).

In the first case, the user simply mounted the camera (and Ludl filter wheel) using the C-mount, but ensured that the camera would not be accidentally kicked by users of the microscope by building their own plastic "cage" around the camera.

In the second case, we designed and supplied a mounting frame that bolted to the underside of the table, although I have to admit it was not fun to install due to needing to sit under the table and work against gravity!

My feeling is that C-mount would be sufficient, as long as you can ensure the camera does not get bumped. If you're worried (e.g. movement of filter wheel could cause wobble), then speak to us or your mechanical department and have a frame of some sort made.

I believe our standard thread on the cameras is 1/4 20.

Hope this helps!

James
________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of Vitaly Boyko [[hidden email]]
Sent: 09 September 2014 02:38
To: [hidden email]
Subject: mounting a heavy Andor camera to a bottom port of a scope

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Dear List,

Would someone advice on the best way to mount a heavy iXon 897 Ultra camera together with a filter wheel to a bottom port a scope. iXon has 8 mounting points, 4 on each side. It could be too heavy for using C-mount only

Thank you,

Vitaly

[Michael Giacomelli]

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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 <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> 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:[hidden email]] On Behalf Of Michael
> Sent: Monday, 1 September 2014 6:34 AM
> To: [hidden email]
> Subject: Fast DAPI staining protocol
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> 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.