Problem with HUVEC - for immunofluorescence (Feb/06/2009 )

Hi, I am new to cell culture.
I was trying to seed HUVEC in a 24-well plates for immunofluorescence (IF). The lab technician told me to put a piece of glass slide in the wells for possible high manification observation under microscope. And, as usually, we need to coat the substrate with gelatin for HUVEC. Then, in this case, I coated the glass slides. However, most of the cells just adhered the edge of the wells or the glass slides,; is seems like they don't like the slides. As HUVEC is monolayer cells, when there are too much cell at the edge, they started to dye while there is no cells on the centre of the slides. As I need to stain the protein in adherent junction of cells, this situation is apparently not good.

I tried many times and failed.

Does anybody here have good suggestion or experience here?

thanks in advance

Hai,

Do you coat the glass slides separately and put it in the well, or add gelatin in the same well and coat. If you are doing in the same well, then there could be coating problems.

We do coat coverslips in the given way:
Coveslips are coated with poly-Lysine overnight in a 50ml falcon tube at 4 degrees. Then sterilized by hot air oven (placed in a glass petri-dish- in between layers of filter paper). Then we store this and use. It works good.

All the best

haiyan on Feb 6 2009, 11:37 AM said:

Hi

Has anyone tried HUVEC culture without coating the flasks or plates???

Confused!!

hema on Feb 24 2009, 11:13 PM said:

CKtong on Mar 6 2009, 04:44 AM said: