Re: [Histonet] Re: overprocessing

From:Esther Peters

Dear John,

Thank you for your insights and I would appreciate getting the references.

We have enrobed specimens in 1.5% low gelling temperature agarose. 
While sometimes we have no problems with our short processing runs, 
sometimes the agarose shrinks and hardens.  I pre-embedded bacteria in 
the agarose to form 1-2 mm-thick disks, then processed and embedded on 
biopsy times.  About half the samples shrank.  I had one cassette 
containing two disks, these were from one that had been sliced in half, 
so otherwise both were treated the same.  One disk shrank and hardened, 
the other one was fine!  So there is a very narrow threshold at which 
the water removal goes "critical."

I have not worked with HistoGel.  Is this a better product for this 
purpose compared to agarose?

Esther Peters, Ph.D.
George Mason University

John Kiernan wrote:

> Dear Carlos (whoever and wherever you are),
> You are quite right, and your observations are
> supported by published literature that has been
> through the academic (= accept nothing without
> proof) peer-review process. 
> Hardness is difficult to evaluate, but anyone can
> measure changes in volume or linear dimensions.
> This was all done decades ago, and I'll be happy
> to send literature references to anyone who's
> seriously interested. 
> Cross-linked polymers 
> (such as the gels used in PAGE) shrink rather
> suddenly when water removal (by evaporation or
> alcohol) hits a critical level. I tried this in
> the lab after reading a Scientific American
> article about 20 years ago. There was a rapid
> change from soft & slimy to hard & gritty, exactly
> as Sci. Am. said it would be. I can't find the ref
> for this pre-computers Sci. Am. article. 
> Shrinkage of animal tissues is less than that of
> highly hydrated polyacrylamide gels or gelatin. A
> linear measurement in a stained and mounted
> paraffin section may be about 70% of the in vivo
> measurement, but the % can vary with the organ. 
> I can send references to anyone who's intersted in 
> following this up. 
> John Kiernan
> London, Canada
> _______________________________
> carlos wrote:
>>Hi Kemlo,
>>I must disagree re hardening......if only in my experience of the following:
>>I have processed gelatin ( 1%)blocks after formalin fixation (  1cm blocks
>>fixed for 2, 5, 10 and 60 days) and find that they all suffer from shrinkage
>>and hardening. My processing is via gradual alcohols to alc/xylene 1:1, then
>>xylene x2 and wax x2. Two hrs in each.
>>Be grateful for enlightenment.
>>Best wishes
>>Histonet mailing list
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