Re: Cutting fixed frozen tissue

From:"C. H."

Hi, Stephanie

Try soaking the brains in 10% sucrose.  30% sucrose is too high a 
concentration for a cryostat and is more appropriate for sliding microtome 
cryosectioning with dry ice.  Alternatively, if you stick with 30% sucrose, 
you could try dropping your cryostat temp. to -30*C...not a great 

Also, do you collect the sections with cold slides? In my opinion, it is 
easier to deal with/avoid wrinkles and distortion by cold mounting as 
opposed to warm mounting.  You have greater flexibility in manipulating the 

I hope this helps.


>From: Stephanie Rodriguez 
>Subject: Cutting fixed frozen tissue
>Date: Thu, 16 Aug 2001 09:18:44 -0700
>Hello!  I am working with a protocol for in-situ hybridization on mouse
>brain that uses the following tissue prep:
>-Whole brain is removed from mouse and immersed in 4*C 4%
>paraformaldehyde and fixed overnight at 4*C.
>-Tissue is then removed from fixative and immersed in 30% sucrose and
>stored at 4*C until it sinks.
>-Brain is then cut on cryostat at 15-20m.
>The problem I am having is that when I cut the tissue and try to mount
>it on slides, it won't lay flat, so it sticks to the slide quickly in
>some places, and more slowly in others, leading to a very wrinkled
>section.  The tissue works great as far as hybridization is concerned:
>we do get a signal; but it looks terrible!  And the signal is
>inconsistent because the tissue is all folded up, so some areas are
>difficult to stain.
>Does anyone out there have any suggestions as to how to mount the tissue
>Thanks for your help,
>Stephanie Rodriguez
>Primal, Inc.
>Seattle, WA

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