[Histonet] Some questions about cryosectioning
gcallis <@t> montana.edu
Tue Aug 10 11:02:17 CDT 2004
I would not want to overfix in Bouins, do not change that step - to keep
your fixation optimal.
Getting rid of Bouins in 70% alcohol puts your tissue in an 'antifreeze'
state with the alcohol (any alcohol in tissue can intefer with proper snap
freezing) so the sucrose cryoprotection step should probably be done with
at least one or two changes to insure the NO alcohol is in the tissue at
snap freezing time, basically you are rehydrating with sucrose in aqueous
solution. If you dissolve the sucrose in PBS, and keep tissues in
refrigerator, you may get away with longer storage - however, snap frozen
tissues in the block are probably the most stable way to store your
tissues. Just store your frozen blocks in a -80C freezer, inside a tube or
ziplock plastic food bag until cryosectioning time.
Sections should probably be air dried and stored in a -80C freezer with
dessicant (16 mesh silica gel in a bag) and brought to RT in an enclosed
box for 30 min prior to immunostaining. Protect your antigen as much as
possible. It will depend on the stability of antigen whether you can store
them at RT before IHC, this is also true of other storage
temperatures. You may have to determine that or someone may know the
answer already. We NEVER store frozen sections at 4C, ideally -80C, or -27C
for a shorter time. One thing we do avoid is any water condensation that
may form on a section going from cold to warm temperature, also never store
the FS inside a cryostat.
OR better yet, do the immunostaining as soon as possible after sectioning.
At 06:42 AM 8/10/2004, you wrote:
>I have some questions about cryosectioning.
>We use the following protocol for brain tissue of xenopus (a kind of frog)
> a.. Bouinn fixation by perfusion, one night postfixation
> b.. Wash in alc 70%, washing away picricacid
> c.. sucrose
> d.. section and mount on glass.
> e.. immunostaining
>Just a normal routine, I think. I am used to do everything in one week, I
>begin with the perfusion and at the end of the week I have my sections
>Now the question: because I have to do many tissue's I cannot handle it
>in one week. The immunostaining has to be done for all tissue's at the
>same time etc..
> a.. What is the best step to leave the tissue in for a while?
> Bouinn, alc 70%, sucrose, and still have good immunostainings and good
> What is for each different step the maximum time, does it mather anyway?
> b.. Already mounted on the slides , how long can you leave them (RT or
> 4C) on the slide and still have good immunoreactivity.
>Dept. Cellular Animal Physiology
>University of Nijmegen
>6525 ED Nijmegen
>frouwke <@t> sci.kun.nl
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