[Histonet] Re: Cryosectioning mouse kidney problems

[Mira Krendel]

Thank you, Belinda and Gayle, for your suggestions.

Sounds like I will need to double-check the temperature at which the sectioning was done.

The tissue is fresh (we used to fix with PFA and then cryoprotect with sucrose but we have switched to using freshly frozen kidneys (snap frozen in OCT) several years ago.  Have not really had any major issues until now so I will need to check whether any settings on the (shared) cryostat have been changed recently.

We use disposable blades; the latest attempt was done with a high profile blade on a different cryostat but with the same issues.

Thanks again,
Mira

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I have questions since mouse kidney is generally easy to cryosection without problems.  
 
You never said whether you prefix with NBF or PFA  or only use fresh tissue prior to snap freezing?   You CANNOT cryoprotect fresh tissue or you will have excessive swelling of the tissue.   Sucrose cryoprotection can only be done with NBF or PFA fixed tissue to prevent large ice crystal freezing artifact.  
 
You also did NOT say what temperature you were using for cryosectioning?   Please clarify. 
 
You also did not indicate what kind of knife or disposable blade you are using?   The sharper the better.    
 
I suspect you are sectioning too cold, so try -20C, the blade and block temperature must be exactly the same temperature.     If you go from the cold e.g. freezing platform on most cryostats directly to sectioning, the tissue will be too cold and chatter when blade passes through tissue.   Cold freezing platforms are often 10 to 20 degrees colder than the desired cutting temperature.   Let the blocks with tissue equilibrate off the platform, near the mictorome itself.   We measure the hot and  cold spots in our cryostat to know where we can equilibrate the tissue to sectioning temperature. 
 
Your blade holder could be loose, the blade angle off a bit and or the blade is getting dull.      
 
High profile disposable blades are superior for cryotomy, more stable and just as sharp as low profile.   If you are using steel knives, do you use a sharp, NEW edge for sectioning.   My hopes are you use disposable blades.   
 
It is important to section the kidney at an angle, so you don't go across the broadest part of the kidney first.   If you do the latter, chatter will occur as the disposable blade meets with resistance that can be felt when blade edge engages the tissue.   Don't cut to thick as  5 um is ideal.  Double check your blade angle/tight screws/sharp blade.    Ahhhh the fine details. 
 
Gayle M. Callis
HTL/HT/MT(ASCP) 

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You will not need sucrose if it is frozen unfixed but it does sound like the tissue froze too fast/ too cold

Try warming the tissue with your thumb just prior to cutting a section and see if that helps

B