[Histonet] Hydrating Frozen Tissues embedded in OCT for Sectioning

[Kim Merriam]

Also, make sure that the block sits in the cryostat for a while to acclimate to the temperature.  The cutting temp for lung (correct me if I'm wrong here) is probably about -20; the block should be the same temperature as the cryostat.


Kim Merriam, MA, HT(ASCP)QIHC
Cambridge, MA


________________________________
From: Colleen Forster <cforster <@t> umn.edu>
To: Kim Merriam <kmerriam2003 <@t> yahoo.com> 
Sent: Monday, February 27, 2012 1:55 PM
Subject: Re: [Histonet] Hydrating Frozen Tissues embedded in OCT for Sectioning

Yep, the sample is too cold. Rubbing your finger across even with a 
glove (just linger a bit longer) will help alot.

Colleen Forster

U of MN


On 2/27/2012 12:47 PM, Kim Merriam wrote:
> As long as you don't need to use them for RNA analysis, the easiest thing to do is just rub your finger (without glove) across the block (very briefly) and then take the section.  This will probably do the trick and hydrate it just enough to make a difference.
>  
> Kim
>
> Kim Merriam, MA, HT(ASCP)QIHC
> Cambridge, MA
>
>
> ________________________________
> From: "Kasai, Miki (NIH/NCI) [E]"<kasaim <@t> mail.nih.gov>
> To: "histonet <@t> lists.utsouthwestern.edu"<histonet <@t> lists.utsouthwestern.edu>
> Sent: Monday, February 27, 2012 1:34 PM
> Subject: [Histonet] Hydrating Frozen Tissues embedded in OCT for Sectioning
>
> We are trouble-shooting cryosectioning mouse lung tissue.  Often the lung
> section tears or breaks apart during sectioning.  In the past, if the lung
> section is proving difficult to section, we take the OCT-embedded tissue and
> re-embed it back into OCT (basically put fresh OCT into the original mold
> and then place the OCT block with the tissue back into the mold such that
> the exposed tissue is covered back with OCT).  This is then placed back in
> our -80°C.  When sectioning the next day, the tissue is often easier to
> section.
>
> One person in our lab tried to resolve the problem by brushing a little bit
> of sterile water onto the tissue when sectioning.  This appeared to hydrate
> the tissue and it sectioned better.  However, we weren't sure if this was a
> good idea or not.  Any feedback would be greatly appreciated.
>
> For background purposes our lung tissue are processed several ways:
>
> 1.  Lungs are perfused with PBS, tissue extracted from mouse, placed in
> PFA/sucrose for several hours and then embedded in OCT.
>
> 2.  Lungs are perfused with PBS, inflated gently with OCT/PBS (3:1), placed
> in PFA/sucrose for several hours and then embedded in OCT.
>
> 3.  Lungs are perfused with PBS, inflated gently with OCT/PBS (3:1),
> embedded in OCT and frozen by immersion into liquid nitrogen (just the
> bottom half of the mold is lowered into LN).
>
> Much appreciation,
> Miki Kasai
> Biologist
> Pediatric Oncology Branch
> NCI, NIH
> CRC, 1W Rm. 1-3-888
> 10 Center Drive
> Bethesda, MD  20892
> (301) 496-2318
>
>
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