[Histonet] Counterstain for fluorescent tissue

[Adam .]

Hi,

The most common counterstains for fluorescent work is the nuclear
counterstain DAPI, which fluoresces in the UV spectrum. If your
microparticles fluoresce in that wavelength, then you obviously can't use
that but there are a whole host of other nuclear counterstains that
fluoresce in pretty much any part of the spectrum you want (see
http://www.cbit.uchc.edu/microscopy_facility/fluorescent_probes.html for the
most common). Various companies sell antifade mounting media with DAPI
included, which is really easy to use. You section your tissue, add the
mounting media right on top of the section, coverslip, and then seal with a
sealant (usually run-of-the-mill nailpolish).

Now here's the problem. Nuclear counterstains stain nuclei really well, but
unlike hematoxylin, you often can't see the cell boundaries that well.
Sometimes you're lucky in that your tissue is a bit autofluorescent and you
can see the cell boundaries that way, and sometimes you can do fancy
microscope and optical tricks such as differential interference contrast
(DIC). If neither of these work, people use antibodies to highlight some
really abundant protein in your tissue such as cytoskeletal proteins.

For your work, there might even be a simpler solution. If your
microparticles survive hematoxylin staining, you can often just take a
bright field photograph with the blue counterstaining, switch on the
fluorescent filters, take photographs of that, and then merge them together
using image processing software such as Photoshop.

Adam

On Sat, Jan 9, 2010 at 12:46 AM, John Kiernan <jkiernan <@t> uwo.ca> wrote:

> Dear Nick Evans,
>
> First:  Say who and where you are, and who is in charge of your experiments
> with mice.
>
> Second:  Tell your boss to buy two or three histotechnology textbooks
> (about $50 each) and allow himself and you and all your colleagues 30 mins
> paid daily reading/lunch time.
>
> John Kiernan
> Anatomy, UWO
> London, Canada
> = = =
> ----- Original Message -----
> From: Nicholas David Evans <ndevans <@t> stanford.edu>
> Date: Friday, January 8, 2010 20:18
> Subject: [Histonet] Counterstain for fluorescent tissue
> To: histonet <@t> lists.utsouthwestern.edu
>
> > Dear all,
> >
> >
> >
> > I am sorry if this is a bit of a basic question. I would like to
> > observelocalization of fluorescent microparticles embedded in
> > mouse skin. I plan
> > to do this simply by cryosectioning skin, mounting the tissue without
> > fixation, and observing under the fluorescence microscope (the
> > particlesare added before killing the mouse).
> >
> >
> >
> > However, I would also like to counterstain the tissue without
> > losing the
> > fluorescence of my sample (so I can see similar features as I
> > might using
> > H&E). Can anyone suggest a good way to do this? I would be very
> > gratefulfor any advice.
> >
> >
> >
> > Many thanks
> >
> > Nick Evans
> >
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