[Histonet] Viable radius (need a chemist!)

From:"Jack England"

Aloha Histonetters,

I'm in a bind and I need some help.  I'm trying to find/develop an assay 
that will determine the radius of viable tissue in a cross-section of FFPE 
tissue that is composed of living cells around a necrotic core.  Standard 
nuclear stains (DAPI, hematoxylins, Sytox, etc) aren't enough, because they 
just show nuclei, and most stains I've looked at won't work because I'd have 
to fix and process the tissue before using them (thus killing all viable 
cells). I don't have access to a cryostat.

My thinking so far is to incubate the tissue with viability reagents before 
fixation, then fix the tissue with the reagents in place.  However, most of 
the mothods I've seen (acridine orange, calcein, etc.) involve reagents that 
are soluble in the solutions that are used in tissue processing.  I thought 
about using a tetrazolium-salt metabolic assay (such as MTT), but since the 
formazan crystals that result are soluble in polar organic solvents (eg, 
ethanol and isopropanol), I'm pretty sure they'd wash out before I even got 
to sectioning.

What I'd love to see is the insoluble crystal formation of a tetrazolium 
salt assay combined with the indiscriminate covalent bonding of a tyramide 
radical, so that my resulting chromagen (which indicates viable cells) won't 
wash out in ethanol, clearant, or water.  Maybe a chomagen-conjugated 
tyramide would work (using endogenous peroxidases to my advantage, over a 
long incubation time)?  Or maybe go with a MTT assay, but dehydrate in dry 
air with a dessicant, rather than a polar solvent (clearing in xylene as 

Can anyone help? I'm feeling like I'm right on the verge of cracking this, 
but doomed to fall just short because my organic chemistry isn't up to 
snuff.  Thanks so much for your help!

--With thanks and aloha,
Jack England
Tissue Genesis, Inc.

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