Neutral red (CI 50040) is an excellent fluorescent Nissl stain: 0.002%, in
water, for 5 minutes; dehydrate, clear, and mount in DPX or another
non-fluorescent resinous medium. With excitation by either near-UV or blue
light (range 325-500nm) the Nissl substance and nuclei fluoresce yellow-orange.
Reference: Allen, D. T. & Kiernan, J. A. 1994. Permeation of proteins from the
blood into peripheral nerves and ganglia. Neuroscience 59(3):755-764.
We used this very dilute neutral red as a fluorescent counterstain for
paraffin and cryostat sections of formaldehyde-fixed tissues from rats that
had received iv injections of rhodamine-labelled albumin (green excitation,
orange-red emission, localization mostly extracellular).
Franz Nissl was a man, not a granule, substance or stain, so we should give
his surname its capital N. Check out http://www.whonamedit.com.
John Kiernan
Anatomy, UWO
London, Canada
= = =
----- Original Message -----
From: TF <ti...@foxmail.com>
Date: Saturday, January 9, 2010 23:19
> How about fluorescent nissl?
>2010-01-10
> TF
> 发件人： Nicholas David Evans
> 发送时间： 2010-01-10 02:16:57
> 收件人： Adam .
> 抄送： histonet
> 主题： Re: [Histonet] Counterstain for fluorescent tissue
>
> Dear Adam,
> Thanks very much for the very helpful advice. I guess my real
> question, which you're answered for me, was - is there a
> histological stain v similar to H&E that isn't fluorescent
> itself, and which you can use without screwing up the intrinsic
> fluorescence of your tissue? I'll just stick to fluorescent
> counterstains as I had originally planned.
> Sorry to offend you John. I had assumed that histonet was a
> useful forum to get advice on histology and not somewhere to
> expect slightly narky, rhetorical responses.
> Best wishes
> Nick
> ----- Original Message -----
> From: "Adam ." <anonwu...@gmail.com>
> To: "John Kiernan" <jkier...@uwo.ca>
> Cc: "Nicholas David Evans" <ndev...@stanford.edu>,
> histo...@lists.utsouthwestern.edusent: Saturday, 9 January, 2010
> 09:41:38 GMT -08:00 US/Canada Pacific
> Subject: Re: [Histonet] Counterstain for fluorescent tissue
> 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 <
> jkier...@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 < ndev...@stanford.edu >
> Date: Friday, January 8, 2010 20:18
> Subject: [Histonet] Counterstain for fluorescent tissue
> To: histonet@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
> >
> > _______________________________________________
> > Histonet mailing list
> > Histonet@lists.utsouthwestern.edu
> > http://lists.utsouthwestern.edu/mailman/listinfo/histonet
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