[Histonet] Re: Mouse brain cryosectioning help

[Donna J Emge]

Hi Mohammad,

For the cracking during cryo sectioning of the mouse brain, that would likely be a temperature issue. Make sure to give the samples just out of a -20 or -80 freezer 15 minutes or longer to adjust to the cryostat temperature before sectioning. Fresh frozen brain typically requires a warmer temperature for quality sections than other tissue types – I section fresh frozen mouse brain at a cryostat temperature around  -17 ᴼC  or -18ᴼ C. You can easily determine if a warmer cryostat temperature is required by placing your thumb on the sample face for exactly two seconds and then taking a section. If the section looks good, then the cryostat temperature needs to be warmer.

For wrinkled sections:  The wrinkling can happen sometimes when cutting 4% PFA, sucrose cryoprotected brain samples.  Flat sections that wrinkle when picked up on the slide.  Using a soft brush, moisten it with distilled water and swipe it across the area of pickup on your slide, then pick up your section on the moistened area of the slide. It will flatten. Works like a charm! You do not need the brush too wet, so shake off the excess water before swiping the slide. Also, if doing insitu hybridization use inactivated DEPC or RNase free water using standard clean techniques for the brush between samples.

4% PFA, sucrose cryoprotected mouse brain samples typically require a colder temperature for sectioning than fresh frozen mouse brain – I section them at minus -20 ᴼC  or -22ᴼ C. You can easily determine if a colder cryostat temperature is required by using a freeze spray and then taking a section or two. If the section looks good, then the cryostat temperature needs to be colder.

If temperature adjustment does not work then other things need to be looked at: fresh sharp blade, knife tilt angle, section pick-up technique, incorrect tissue freezing technique…

Stay tuned: Soon I will have another very detailed You Tube presentation on tissue freezing techniques and frozen sectioning tips for fresh tissue, fixed sucrose cryoprotected tissue, and for enzyme studies.

Donna

Donna J. Emge, HT-ASCP
Mouse Histology and Phenotyping Core Manager
Northwestern University
Olson Building room 8-333
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Chicago, IL  60611
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