Re: cryostat

From:Geoff McAuliffe

Hi Jocelyn:

Jocelyn Torcolini wrote:

Hi Histonetters,

I have a few questions about cryostat sectioning.  

In what situations is a cryostat better than regular paraffin processing, and why? 

Faster to get the sections under the microscope and
lipids are not lost with dehydration and clearing and
some immunoreactivity may be lost with fixation (depending on the antigen).
However, morphology is worse with fresh frozen sections and
some enzymes and antigens may diffuse without fixation giving less precise localization.
So "better" is relative to what you want to do.

How do you get cryostat sections to stick to a slide for staining?

In my (limited) experience the proteins from tissue fluids allow for good adhesion even on plain glass slides. Your mileage may vary. For long proceedures I would use coated (gelatin, silane) slides.

Do you need to fix tissue for cryostat in paraformaldhyde/sucrose solution?

You can, depending on the goal. You can also fix tissue first, then freeze and cut on a cryostat or a sliding microtome. The latter gives much better morphology.

 Can you section fresh/frozen tissue?

Yes. You can section fresh, unfrozen tissue down to about 20 microns with an instrument called a Vibratome. This does take some practice. For thicker sections you can use a "Stadie-Riggs" tissue slicer.
You can freeze fresh tissue with dry ice or liquid nitrogen and section it with a cryostat.


Thanks in advance,


Jocelyn Torcolini

Electron Microscopy Facility

1 Frear South Building

University Park , Pa 16802


Phone (814) 865-0212


Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
675 Hoes Lane, Piscataway, NJ 08854
voice: (732)-235-4583; fax: -4029

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