[Histonet] Re: 70% from NBF

From:Gayle Callis

Joseph made some excellent points here

Chloroform is an excellent clearing agent (used it back in the 60's in open 
dip and dunk processors - O.K. so I'm old!) but no one warned us about its 
carcinogenic nature and there were no safety issue regulations then.  Take 
his advice!   Clearite 3 and Propar, two xylene substitutes we have used, 
do not harden tissues excessively but they are sensitive to water.  Careful 
monitoring of how many tissues pass through substitutes is advisable then 
either rotate and/or change these more frequently.   On our VIP, we have 
one xylene followed by one Clearite 3 to help reduce hardening while giving 
better tolerance level/removal of water by xylene in first step.

We work with rodents and 70% alcohol storage after NBF fixation and a brief 
rinse presents no problems.  We do store brain and spinal cord in 50% 
alcohol but all processing starts in 70%.  We use custom processing 
schedules for brain with shorter schedule smaller  tissues, sliced brain 
and spinal cord and/or tissues that tend to be brittle after over exposure 
(a.k.a. over processing) to alcohols, clearing agents and heat of 
paraffins.  Heat is never added to any tissue processing of animal tissues 
especially murine.  Animal tissues are very lean and any extra heat dries 
out the tissues even more. Oh wise one,  Jerry Fredenburgh (in a panel 
discussion) said you want to remove free water from the tissue spaces but 
not the water bound to the proteins as the latter contributes to hard, 
brittle tissues at microtomy.

Acid decalcified bone is not harmed by 70% alcohol storage, in fact, this 
is a way  to stop acid decalcificaton (calcium does not ionize in presence 
of alcohol) and prevent over exposure to acid (aka over decalcification).

Gayle Callis HTL, HT, MT(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University
Bozeman MT 59717

Although At 11:27 AM 11/28/2005, you wrote:
>Lorraine, going from NBF to 70 is very common.  What you are doing is 
>perhaps overtreating the samples in alcohol.  Of all specimens spleen, 
>pancreas are really susceptible to dehydrations negative effects.  If you 
>are using chloroform, my fitst question is why?  You would want to 
>consider Chlorofrm as a dehydrant as well as a clearant, but get rid of 
>that stuff.  Just go fromt NBF inot to 50 or 70, do the washes if you need 
>and cut down on the time or heat.  The samples that have been done should 
>just be soaked.  You will note in Freidas book she states that 70 is the 
>highest alcohol you can go into from NBF(unwashed), anything higher and 
>you risk the buffer salts not dissolving.  I am lazy, I do not wash after 
>formalin since I use it on the processor.  I go into about a 60% alcohol, 
>and move on.

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