mouse kidney fixation - (Dec/23/2009 )

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

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...

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

-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.