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Author Topic: shrinkage  (Read 2195 times)

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Offline peterex

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shrinkage
« on: January 04, 2008, 05:00:32 PM »
I transcardially perfuse with 4% paraformaldehyde in phosphate buffer, then gradual penetration to 30% sucrose in PBS (2 or 3 days at 4C).  The whole brain is then stored at -80 until sectioning, and mounted directly on the chuck with OCT.  My problem is shrinkage of tissue during some step, most likely freezing.  When the brain is sectioned, bubbles and wrinkles of the tissue can not be avoided when they stick to the slides.  When a section (20 um) is floated in PBS, it immediately expands to twice the size, suggesting some osmolality issue.  Any suggestions out there?

shrinkage
« on: January 04, 2008, 05:00:32 PM »

Offline richard03

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Re: shrinkage
« Reply #1 on: January 06, 2008, 01:00:22 PM »
The brain may be under fixed. Are you sure the perfusion went well? Even though you will probably still need to postfix for a few hours before transfer to sucrose. It would be best if the brain went through low conc of sucrose first, i.e. 15% sucrose until it sinks to bottom and then 30% sucrose until it sinks to bottom.

Freezing step is also critical and it need to be rapid (~30 seconds). What did you use for freezing brain tissue?

 

Offline peterex

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Re: shrinkage
« Reply #2 on: January 07, 2008, 01:54:10 PM »
There's always a concern of whether there's sufficient fixation, as we don't postfix at all.  After perfusion, the brain is removed immediately (to prevent over-fixation) to 15% sucrose in PBS until it sinks (overnight), then to 30% until it sinks.  The whole brain is burried under crushed dry ice for about 2 minutes, then stored at -80. 


Offline richard03

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Re: shrinkage
« Reply #3 on: January 07, 2008, 06:42:20 PM »
You might get freezing artifacts as you were using dry ice for 2 minutes. It was too long. You may consider to use isopentane cold in dry ice or liquid nitrogen also be better than dry ice.

After freezing the brain, it needs a layer of ice covering the frozen brain to protect it from drying. You can do this by putting drops of water on the surface of frozen brain in Cryostat or -80 C freezer, repeat this more time to get thicker protection. Then you are safe to store the brain blocks in -80 C.

Re: shrinkage
« Reply #3 on: January 07, 2008, 06:42:20 PM »