Author Topic: Frozen sectioning unfixed tissues that are destined for -80 storage  (Read 1969 times)

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Offline lucieg

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It's been a million years since I last used a cryostat and for the life of me I can't remember how to deal with my slides while sectioning.

I'm cutting fresh frozen tissue that is intended to go straight back into the -80 freezer as soon as the sectioning is completed (no fixation). Is it ok to temporarily (minutes, not hours) keep the slide at room temperature with sections already on it while I cut the next section(s)? This obviously means that the already cut sections will be thawing and beginning to dry before the sectioning is completed and the slide is eventually thrown back into the -80.

The plan is to have 3 sections each from 6 different blocks on each slide. Should I keep the slide at RT while collecting the 3 sections, then back into the cryostat while I set up the next block, then back to RT while sectioning, etc.? Or am I being too paranoid and leaving them to thaw and dry at room temperature before going back into the -80 is standard protocol?

Thanks a bunch!


Offline CanuckPhD

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Re: Frozen sectioning unfixed tissues that are destined for -80 storage
« Reply #1 on: October 26, 2011, 02:44:08 AM »
I cut my sections, leave at room temperature to dry for 30 to 60 minutes (depends on how many sections I need to cut) and then put in the -80 for storage. To date I don't have any issues with my antibodies (mainly markers for neurons, glia and immune cells). However, if the tissues are to be used for LCM, ISH or something more sensitive then you will have to be more careful about thawing.

Also if you don't let the sections thaw and dry for a short time they do not adhere to the slide and you will have a lot of tissue loss.

Offline GWinn

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Re: Frozen sectioning unfixed tissues that are destined for -80 storage
« Reply #2 on: November 16, 2011, 06:52:25 AM »
Hi - I think Canuck is right. We cut slides as quickly as possible, allow to dry the store the sides in a sealable container with dessicant until use.  The biggest problem that Ive seen is the frosting of slides prior to asy fixing after storage.  So if you are keeping unfixed slides in the freezer - you need to pretty much go from freezer to fix without getting frost on them. The frost will cause artifact and likely damage antigenicity. Sure, you have to pick up slides for each section (on a "warm slide") but a cold slide may mean both bad adhesion and frost=moisture=ice on the sections.
Regards

Re: Frozen sectioning unfixed tissues that are destined for -80 storage
« Reply #2 on: November 16, 2011, 06:52:25 AM »