Hi,

1- Try to seed more cells / well.

2- If your cells have a problems in the attachment to the coverslip (try to treat the coverslips with collagen-I overnight to increase the attachment of the cells).

3- Prepare fresh PFA 4% and increase the time of fixation to 15 min.

4- You can use methanol for fixation also, it works better for specific cell lines.

Best

Washing could be the problem. Do not throw any solution directly on the cells. go from one side very gently. Also, you are using PBS alone. Washing solution need to be PBST with some serum or BSA, that helps too. Treat gently the cells even if you are in a hurry.

Try Yaser's suggestion also

Hi,

Yes, most likely it is wash steps that are taking away your cells from the coverslips. Make sure that you add the PBS gently through the wall and not directly on the cells. Some of the cells are anyhow less adherent to the coverslip, if this is the case, you can pre-coat your coverslips with something like Poly-L-Lysine. Also, make sure that you are using the correct size of coverslips which fit very good inside the wells (e.g. if you put too small coverslips inside 6 well plates during the washing step on the shaker they are moving too much).

Last idea: before starting the staining have a look at the cells and make sure that you have enough cells, not that from the beginning you have a very low density of cells.

Hope this could help and good luck! Behnam