Thank you for your valuable answer.

I agree with Sebastian's comment above, although I've always been told to use cold PFA, as the free formaldehyde (i.e. the molecule that actually acts as a crosslinker and therefore a fixative) tends to polymerise at higher temperatures, therefore becoming less effective.

The method I generally use for ICC (on different cell lines, although not KYSE-30) is the following:

The cells were then washed with PBS and fixed with 4% v/v paraformaldehyde/PBS for 30 minutes. For immunocytochemistry and phalloidin staining, permeabilization was performed using 0.25% v/v Triton X-100/PBS (Sigma-Aldrich) for 30 minutes followed by blocking using 5% v/v goat serum/PBS (Sigma-Aldrich) for 1 hour.

Make sure to carry out enough washes (e.g. 3x 5min in cold PBS) between each step.

With ICC, as in the case of Western Blots, a lot of the end results depends on trial and error. Before you start your actual experiment, I recommend to process some samples in parallel by using different conditions and see which one(s) work better with your specific cells/antibodies.

As Sebastian said, 4% PFA followed by Triton x-100 (generally 0.1-0.25%) is pretty much standard procedure, however some antibodies might require a different fixative or blocking solution, and you pretty much won't know it until you try!

Good to know, I'll have to try it out!

Thanks a lot for your comprehensive information. Could you please let me know what is the best way to attach these cells to a glass slide for ICC? I mean

what cell count is necessary for each glass slide and also what is the best program of cytospin for this sample?

Although it might be a bit more expensive than conventional slides, I strongly suggest using a "slideflask". It's basically a small tissue culture flask with a microscopy-ready bottom. You can just grow the cells in there as normal, then "pop it open", and you'll be left with a slide ready to fix and image. Here's a link to that product https://www.thermofisher.com/order/catalog/product/170920 .

In terms of cell number, it really depends on the cell type, and what kind of analysis you want to do. Are those cells contact-dependent? Do they like high or low confluency? Once you know the desired count, you can just resuspend your initial pellet, dilute it as appropriate, and seed the appropriate number in a slideflask (or glass slide).

I agree with Alessandro about growing your cells on a 'slide flask'. However, you could grow the cells on a multi chamber pre-coated slide. The multi-chamber slides come with 2, 4, 6 or 8 chambers, thereby allowing to titrate an antibody of choice at several concentration simultaneously. They are readily available at VWR or Fisher Scientific. Following the immunostaining, the upper chamber could be easily peel off from the slide for putting cover slip.

Fixing cell lines or tissues with 10% buffered formalin at ambient temperature is an universal method of choice.

You can fix and permeabilise cells at the same time, using Zenker's fixative (doi:10.1007/BF02623886). The disadvantage is that this fixative contains mercury.

Thank you all for your helpful responses.