Why do you want to stain cells with all markers all at once!

Is it not possible to stain them one at a time using ICC and demonstrating % positivity?

If you want to stain with multiple markers I would try both methods - staining intracellular markers followed by extracellular markers or vise versa

However the disadvantage of staining with multiple antibodies is,

1. Multiple antibody titrations required

2. When you permeabilize your cells, surface antigen expression would most likely be adversely impacted

Suggest you try one staining at a time using ICC

I guess you need the stainings for a characterisation so this would be a qualitive staining. In our lab we use three different antibodys at the same time and it's working fine. I guess it is hardly possible to do more than three caused in the wavelength of the secondary antibodys you'll have to use. (If they are too close together they could overlap!)

So I would reccomend to prepare multiple coverslips and do one double and one triple staining.

Lukas

Immunfluorescence or ICC with colour-developement?

For double-staining it is favourable to choose antigens on different locations. Combine one for the cytoplasma and one for membrane. If, for example, the membrane-antigens are located on different cell-types, this can also bring impressive results to stain them on one slide.

I have only experience with IHC with colour-developement. There it is not favourable to stain colocated antigens in one step. But one can combine antigens on different locations even in one-colour (as done in antibody-cocktails).

Most double-staining commercial kits are sequential, so you do your membrane marker first with DAB and then follow with your cytoplasmic marker. Leica produce a simultaneous double-stain kit called Chromoplex which requires one of the antibodies to be mouse and the other to be rabbit. it may or may not suit your purpose.

Kind regards,

                           Kieran