[Histonet] Counterstain for fluorescent tissue

[Nicholas David Evans]

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.edu
Sent: 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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