Culturing/Mounting of polarised cells for confocal microscopy - (Apr/04/2009 )

Hi there

I have been doing some work on CACO2 and MDCK cells, looking at the localisation of a protein relative to tight junctions in these polarised cells using confocal microscopy.

However, no matter how long I grow them, and at what confluency, although i do get tight junction staining (i.e. a band of ZO-1 tight junction marker staining near the top of the cells), the cells are not that "thick", being only a couple of micrometres thick on the Z axis.

I have looked everywhere to find out how thick these cells should be when polarised but cannot find any information. I fear it may be the way I mount the coverslips as I just fix and mount them like non-polarised cells (HeLa, etc) and think that when i place them on slides I must be squashing them flat!

Any help much appreciated!!!

Chris

Chris,

There's a trick that I learned mounting whole-mount zebrafish embryos (also very squishable) - try placing one coverslip on either side of where you'll place your cells (you can stick them on with high-vacuum grease or on top of a thin coat of nail polish or similar). Make sure the distance between the coverslips is less than the length of the coverslip you're going to place on top so it can bridge them. Place your cells in the middle. Dab a tiny bit of grease on top of the coverslips then put the "bridge" coverslip on top. ... If this leaves too much space under your "bridge" coverslip, try just putting a little grease on either end of the slide then placing the "bridge" directly on top of the grease. A thin layer of grease will hold the coverslip a good deal less than 1mm off the slide.

-Carlton