staining cells on transwell inserts - fluorescence microscopy (Dec/05/2007 )

Dear bioforumers,

I have some protocols for staining cells on a slide. I would like to transpose this protocole for staining cells on a transwell membrane.

The cells are grown on a coated PET membrane, in a transwell insert, in a 24 wells plate.
I thought I could fix the cells in the well, by adding enough volume of PFA in the bottom of the well, and on the membrane, and wash.

Then, to spare antibodies, I thought I could maybe cut the membrane, and put the membrane on a slide or whatever, and process as with coated slides.
(few microlitres of antibodies on a parafilm and put the slide on the antibody drop on the parafilm. If I put antibodies in the well, I will need to use a big volume)

Did one of you already did something like that? Any advice?

Thanks in advance

Missele

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Hi,
Yep, did transwell before, but I always add Ab inside the well. How big is your transwell? I found for me the 24-well (in the catalogue it is 12-well plate, but actually is 24-well with 12-well transwell) is enough. Just add Ab inside the well, not outside, that is not too much Ab.

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Thanks Almasy,

Actually I was hurry up, so I tried.
I put the transwell on a parafilm, and added the antibody in the transwell.
(I tried to cut the membrane before the antibody incubation, but it was a mess. It's difficult to manipulate).

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Well, yes, if you cut out the membrane before incubation, it could be a real mess to work with later. I only peel off the membrane for mounting, although in theory (maybe if you are very careful, in practice, too), cutting out the membrane and then do staining should be possible.

Hope your exp. works out well.

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