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mouse kidney fixation


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#1 mouseIHC

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Posted 23 December 2009 - 10:21 AM

I am seeing collapsed tubular lumens on my mouse kidney sections, and have been unable to resolve the issue.

I am using perfusion-fixation with 4% PFA, with good blanching of the kidneys, cutting the kidneys into 3mm sections and incubating in 30% sucrose overnight. The specimens are then frozen in OCT in liquid nitrogen the next day, then cryosectioned at -22C at 2um sections the day after.

The tissue morphology overall looks well, with back-to-back tubules, patent vessels and open glomeruli. However, the tubular lumens are mostly closed/collapsed, which is not what I am seeing in other publications. This is a problem, because I am trying do IF to stain for a brush border protein, and other papers have shown widely patent tubular lumens and good staining.

Any thoughts would be appreciated!

Thanks

#2 Jojje

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Posted 26 December 2009 - 06:44 AM

Does other people at your lab get nice sections of different tissues, or is it only you that experience this problem? When I encounter problems with getting nice sections it's mostly some problem with the sectioning device, not the protocol used...

#3 Carlton H

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Posted 30 December 2009 - 02:12 PM

Sorry if this seems obvious, but have you changed the blade recently?
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#4 mouseIHC

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Posted 03 January 2010 - 12:57 AM

-unfortunately we are the only ones in the lab doing kidneys. others are doing mouse lungs and are not having any problems.

-yes, I tried using a brand new blade as well.




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