For finding the non-specific binding, you need to have proper controls.

1. Unstained control - which gives information about autofluorescence and you can also adjust the autofluorescence intensity to minimum for proper detection

2. only secondary antibody control - if you are using unlabelled primary antibody and detection with a secondary antibody, It is quite necessary to have this control which tells about the non-specific binding of your secondary antibody.

3. Other cell controls - if the problems arise from the primary antibody, you need to have other cells as controls, in which the protein you are detecting is not expressing. However, it is difficult to find such kind of controls and it is based on what you are detecting.

To avoid non-specific binding, you need to have proper blocking sometimes with different kinds of serums or increased BSA concentration etc.

I hope this information is helpful. More details needed to understand your problem properly.

For finding the non-specific binding, you need to have proper controls.

1. Unstained control - which gives information about autofluorescence and you can also adjust the autofluorescence intensity to minimum for proper detection

2. only secondary antibody control - if you are using unlabelled primary antibody and detection with a secondary antibody, It is quite necessary to have this control which tells about the non-specific binding of your secondary antibody.

3. Other cell controls - if the problems arise from the primary antibody, you need to have other cells as controls, in which the protein you are detecting is not expressing. However, it is difficult to find such kind of controls and it is based on what you are detecting.

To avoid non-specific binding, you need to have proper blocking sometimes with different kinds of serums or increased BSA concentration etc.

I hope this information is helpful. More details needed to understand your problem properly.

If this protein is crucial to your work, I would suggest:

1)  Try a couple of validated antibodies for your protein of interest.  If the staining pattern is consistent, then the signal is likely specific.

2)  Try a couple different fixation methods as these can severely affect your staining.  I'll often try 3% PFA or 100% MeOH the first time I use an antibody.  

3)  Finally, the gold standard, in my opinion, is to knock out or knock down your protein of interest.  If the staining is specific, you should see the signal disappear.  

Good luck!

I suggest an absorption specific control for the samples. You will need pure antigens(preferably synthetic proteins), to absorb/exhaust the antibody for the designated target (your protein of interest). Then use the absorbed/exhausted solution for IFC. If a positive staining is detected, you are confirmed for non-specific bindings (this will apply to both primary and secondary antibodies).

As long as you do not see any immunostaining or lower intensity of immunostaining in (No primary antibody but secondary antibody) control, your staining is most probably real.

Always have controls for both your coculture cells as well as mono-culture cells where you do not add primary antibody but still add secondary antibody to monitor change in intensity of florescence between your test sample (where you add primary as well as secondary) and control sample (where you don't add primary but add secondary). 

Also, taking a ratio of coculture cells and mono-cells is not a great idea as higher number of cells may effect the cellular shape in 2D culture and thus will affect the fluorescence intensity.

First thing you need to make sure is

1) Cellular area of mono cells does not change in your mono-culture as well as co-culture.

2) Number of mono cells also remains approximately same across your mono as well co-culture.

Once these two conditions are fulfilled you may be able to take the ratio of florescence in your co-culture and mono-culture condition to measure changes in expression of protein of interest.

I will agree with all go for the proper control.

Use no primary control to ensure that only secondary antibody is not giving background. Image both the co-culture and monolayer cells carefully with no primary control. Try to keep the intensity low to avoid background due to auto-flourescence.  

You can separately stain the monolayer and co-culture to rule out the background.

Try to use right dilution of the antibodies.

Check your abs quality using western blotting on cellular isolates. it is painstaiking sometimes, mightbe you do not even get signal. you need to detect the proper MW signal, if everything OK: and nothing else.

I have to disagree somewhat and respectfully with Attila.  Standard SDS-PAGE will denature proteins, which will obliterate epitopes that depend on the 3-dimensional conformation of the protein.  These same epitopes are preserved with PFA.  Thus western analysis may not be a good indicator of an antibody's specificity when used for immunofluorescence.  

That is true, Michael.  Wb is not always good. However, conformational epitops are rear nowadays, as most of the abs , even monoclonals, generated using selected short sequence of the target protein.

Checking the specificity by western blot and comparing with staining depends upon the antibody binding epitope. If an antibody is suitable for western blot (under denaturing condition) as well as staining, it is immaterial whether, the protein is denature  or intact. In this situation we can compare the results of staining and WB and comment on the specificity depending upon the band at the right position. If an antibody is conformation specific, staining specific and cannot be used under denaturing condition,  in that case WB cannot be used as a method to define specificity.

Antibody information and its application is crucial.

Hi, 

What type of cells do you culture? If it's, for example, mouse MEFs and human ESCs?

Find the sequence of the proteins which you check coding by your co-cultured cells.

Find immunogen sequence which was used to generate primary antibodies anti your analyzed protein.

Compare sequence of immunostained protein and immunogen.  Immunogen should be designed in the place of the proteins sequences were compared proteins sequence are different for co-cultured cells.

Find new more specific primary antibodies or order primary antibodies production specific for your immunogen.

If you for example culture human astrocytes with human neurons make double staining markers of astrocytes (GFAP) and neurons(MAP2).

Good luck!