Re: gluteraldehyde fixation

From:Geoff McAuliffe

Hi Loarlee:

Gehan, Loralee wrote:

>             I need some help with glut fixation.  My experience with
>gluteraldehyde is that it is a very very slow fix and it needs to be

    Glutaraldehyde fixes rapidly but penetrates tissue slowly. It is 
often used at room temperature, if used cold microtubules are disrupted 
and fixation is slowed.

> Also I haven't fixed anything in it for more than 24 hours
>and the specimens were very small.  Researchers in my lab are insisting that
>I fix their very large specimens (adult rabbit spine) in .25% glut in pbs
>for about five days.  Then EDTA decal (10%) for two weeks.  I then paraffin
>embed these samples or cut frozen sections.   They insist that this will
>preserve lac Z enzyme.  
    It is their project so fix the tissue exactly the way they want. If 
you make any changes and things don't work out, it will be all your 
fault. If the tissue is fixed by perfusion the deeper parts of the 
tissue may be fixed well enough for light microscopic analysis.
    I suspect that they are simply using a protocol they read in another 
paper, fixing for such a long time with such a low concentration of 
glut. does not make much sense to me.

>              What are your opinions on this?  All of my experience tells me
>that the inner part of the spine will not fix before necrosis sets in.   Has
>anyone every done this?  If you have what was your protocol for fixation?
>Thanks all in advance. 
>Loralee Gehan
>University of Rochester

Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
675 Hoes Lane, Piscataway, NJ 08854
voice: (732)-235-4583; fax: -4029

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