Bone for Re: [Histonet] fixation in 70% alcohol

Gayle Callis gcallis <@t>
Thu Dec 8 11:37:16 CST 2005

70% alcohol has been a historically popular fixative for undecalcified bone 
destined for methyl methacrylate i.e.plastic embedding  to help preserve 
water soluble fluorescent tetracycline labeling.

So here are two questions

Are you labeling the bones with tetracycline?

Are you embedding in PMMA or paraffin?

In general, 70% alcohol is a poor fixative for bone!!!  Morphology of cells 
and soft tissues is bad  compared to NBF fixation. If you planning to 
decalcify and  embed in paraffin, it would be advisable to fix in neutral 
buffered formalin.  In this case,  alcohol slows down and can actually stop 
ionization of calcium until the bone rehydrates.

If you are planning to PMMA embed, you will have superior morphology is you 
fix in
         1.  Neutral buffered formalin for general staining without tetracyline
         2.  Alcoholic formalin which preserves the tetracyline label, but 
gives you better morphology than 70%.  One 
recommendation,            Anatech has an excellent alcoholic formalin 
fixative if you don't want to mess with making it up inhouse.

After fixation with NBF, you can store bone in 70% alcohol.

  If your bones are whole, fix rat, mice at least a week or more, and 
change the fixative - this also goes for NBF or alcoholic formalin.   Sheep 
- cut slabs or open bone and fix a longer, it may be up to two weeks for 
whole big bones and maybe even longer if the bones are huge - have done 
sheep and know how big their bones really are!!!!   You fix according to 
size of bone, less time for small, more for large and there is NO 
established time as it depends on the bone cortical versus mostly 
trabecular, removal of soft tissues, size of bone, age of animal, etc.  If 
you open a bone after it has been in fixative and it is still reddish 
inside, you have not fixed long enough.

Good luck

At 09:18 AM 12/8/2005, you wrote:
>70% ethanol is not considered a fixative.  Ethanol fixes by dehydration, 
>so at a minimum you should be using 95% ethanol, and preferably 
>absolute.  70% can be used as a preservative after fixation.  You have 
>unfixed tissue if they are put into it immediately after harvest.  Since 
>it is not a fixative, the length of time is not really a factor.  What is 
>most likely happening is that you then dehydrate with higher 
>concentrations of ethanol and the tissue is fixed at that time.  Ethanol 
>alone is considered to be a very poor fixative, although it may be 
>necessary depending on your application.  I suggest you either use a 
>proper fixative such as a formalin variant, preferably overnight, or put 
>your tissue directly into absolute ethanol for a few hours.
>Bryan Llewellyn
>----- Original Message ----- From: "Fawn Jones" <fawn <@t>>
>To: <histonet <@t>>
>Sent: Thursday, December 08, 2005 7:03 AM
>Subject: [Histonet] fixation in 70% alcohol
>>I just started working in a new lab and they use 70% Ethanol for 
>>fixation, however I do not know how long to keep the tissues in 70% to 
>>ensure adequate fixation. I am working with animal bones (mice, rats, 
>>sheep). Could anyone give me suggestions on fixation times?
>>Thank you
>>Fawn Jones

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367
406 994-4303 (FAX)

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