Re: [Histonet] free floating sections

From:Maria Mejia

Hello Atoska,

I section serial sections of monkey & rat brain on the cryostat @  
40um. I collect the sections using
24 plate wells containing a solution called "anti-freeze." Each well  
has about 2300ul per well (don't over-
fill) This working anti-freeze solution contains:
ethylene glycol - 120ml
gycerol - 120
working PBS (I can't remember the correct amount - will email this  
information when I get back to the lab on
Tuesday). When mixing this solution - mix for at least 30 minutes and  
store in refrig @ 4C. Solution is stable.

Anyway, when I use the cryostat to cut  brain tissue, I keep a small  
glass beaker (50ml size) inside the cryostat
with about 20-30ml of anti-freezer solution. Before I start  
sectioning, I dip my sable brush into this solution and
brush the knife surface very close to the edge w/some anti-freeze  
solution, not a lot, but just enough so that when
I do take a section it slides on the knife. I then use my brush moist  
with anti-freeze to pick-up the section and
place into a plate well until each well has a section. Repeat until  
an X number of plate well are used for each case.
We use 5 to 6 plate wells (one plate has 24 wells) per monkey case.

When the case has been completely section, each plate well (each has  
ID info) is then sealed with cryo-tape.
The 5 or 6 plates are then stacked on top of each other and wrapped  
in aluminum foil with ID info., and sealed
with more cryo-tape. The plates are then placed inside a large enough  
plastic box w/lid & ID to hold more than
one case. Box with plate wells are kept in a refrig @ 4C. They will  
keep for several years and if sealed properly
this solution will not evaporated. When ever we need to do some  
histochemical and/or IHC staining, we simply
remove the sections of interests and wash the sections in 3 changes  
of working PBS - 5 minutes each and
proceed with the staining.

I hope this helps and on Tuesday I will email you the complete recipe  
for making anti-freeze solution.

P.S. When I finish using the cryostat, I simply wipe any residual  
anti-freeze from the knife holder & knife using
kim-wipes with 70% alcohol and then 100% alcohol.


Maria Bartola Mejia
University of California San Francisco
Department of Neurosurgery
San Francisco, CA 94103

On May 26, 2006, at 9:28 AM, Atoska S. Gentry wrote:

> Hello, will someone please share with me your procedure for  
> collecting & storing free floating frozen sections? This will be a  
> first for me. Info gathered from histonet archives indicates the  
> sections can be placed into cell culture plates with wells  
> containing distilled water.  Is distilled water required or will  
> filtered deionized water be sufficient or better yet is it o.k. to  
> place phosphate buffer into the wells? Also, please what's the  
> maximum amount of time these free floating sections can be held at  
> 4C in the culture plates before staining or whatever? I will be  
> sectioning these sections for a researcher from main campus and I  
> need to inform him. Thanks so much. Atoska
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