3 replies to this topic

[mouseIHC]

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

[Jojje]

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...

[Carlton H]

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

[mouseIHC]

-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.