[Histonet] Counterstain for fluorescent tissue

Mon Jan 11 00:38:00 CST 2010

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 <tifei <@t> 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 ." <anonwums1 <@t> gmail.com>
> To: "John Kiernan" <jkiernan <@t> uwo.ca>
> Cc: "Nicholas David Evans" <ndevans <@t> stanford.edu>, 
> histonet <@t> 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 < 
> 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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> > Histonet <@t> lists.utsouthwestern.edu 
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