Re[2]: strange request...

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From:"Jeff Crews"<>
To:<>, <>
Content-Type:text/plain; charset=US-ASCII

     True, most gels don't stain well with eosin (which should stain 
     collagen) just because they are usually mostly water and don't come 
     anywhere near the density of native connective tissue collagen. 
        An hematoxylin with acetic acid should be specific enough to just 
     stain the nuclei and will have more contrast than nuclear fast red.
        And yes, you can image through a gel at least 1mm thick, with a 
     conventional microscope. It's not the clearest image in the world, but 
     you can see what you're looking at and identify cells as long as 
     they're not too tightly packed. Or check your sofa cushions for loose 
     change and buy a confocal system. ;-)
     Jeffrey Crews, HTL (ASCP)
     Organogenesis, Inc.

______________________________ Reply Separator _________________________________
Subject: Re: strange request... 
Author:  denise M m Long-Woodward <> at internet
Date:    04/24/2000 9:18 PM

It has been my experience with skin cell cultures that the 
collagen/fibrin gel doesn't stain with either H or E.
Why not process just one gel (without cell cultures) to verify? 
Denise Long Woodward
On Mon, 24 Apr 2000 16:45:22 -0400 "Johnson, Jennifer(Hist)" 
<> writes:
> Dear Histonetters,
> 	An investigator came to me today with a strange request.  
> She is
> growing small pieces of mouse aortas in a collagen and fibrin gel.  
> She
> would like to be able to stain the aorta and the cells growing from 
> it,
> without staining the gel.  She wants to perform the stain on the 
> whole 1-2
> mm thick plug without sectioning it in order to try and count the 
> number of
> cells that have grown off of the aorta.  I have suggested that 
> processing
> into paraffin may give her more success, but she is really looking 
> for a
> quick and dirty instant answer.  (Doesn't this sound familiar?)   
> 	Since, ideally, the method chosen should be relatively 
> simple, I am
> going to first try a nuclear fast red, or maybe a hematoxylin 
> (without
> eosin), or even a DAPI stain.  My thinking is to stain the nuclei, 
> and avoid
> cytoplasm (I think that in these cells the cytoplasm and aorta 
> itself will
> have collagen in them).  
> 	My second idea is to try a fluorescently labeled S-100 
> antibody,
> (using the most abbreviated protocol I can find!) to label the cells 
> only.
> 	But I am having a lot of trouble finding any other methods.  
> Most
> books are organized by what you want to stain, not by what you are 
> trying to
> avoid staining!   If anyone has had any experience with this sort of 
> staining, or has any ideas, I would appreciate hearing from you!  
> Thanks,
> Jennifer Johnson
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