Re: TEM sample preparation for knee joints
|From:||Gayle Callis <firstname.lastname@example.org>|
For good TEM, really tiny pieces must be used, and EDTA is the
decalcification of choice. Our EM tech just decalcified in approx 10%
disodium EDTA at pH 7.2-7.4, in a suitable buffer for EM (Sorenson's) for
two weeks with several changes and gentle agitation, then extended
processing times due to tissue density with longer infiltrations of resins
before embedding. To remove EDTA, extra washing in the same buffer. If
you use any other decalcifying agent, an acid, ultrastructure will be
damaged for TEM. Dickson's book, Calcified Tissue Preparation has whole
chapters devoted to EM preparation of bone, unfortunately, it is out of
print, unless your university library has it available, or try and get it
on interlibrary loan (from Univeristy of Texas!). Citric acid has been also
used for bone EM work.
It may be advisable to remove articular cartilage from the bone surface,
mince it into tiny pieces in cold buffer fixative on dental wax (a proper
EM fixative) and avoid decalcification altogether, then process it. Your
diamond knife and/or glass knife will be much happier if you do this. Bone
unfortunately is not the rapid, quick handling of normal tissue.
At 01:43 PM 11/17/00 -0800, you wrote:
>I am a college student involved in some research work in which I prepare
>mice knee joints for TEM then look at the ultrastructure of the articular
>cartilage. Currently I use a 3 day standard procedure, but I have heard of
>a microwave prep that could cut the prep time down some keeping the
>structure fresher. Does anyone know any procedure for that? Also what
>would be the optimal way to decalcify the bone to make sectioning easier?
>I appreciate all suggestions! Thank you!
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Veterinary Molecular Biology
Montana State University
Bozeman MT 59717-3610
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