in my experience you do not really need to multiple and separate steps and incubations. Just do a regular IHC with PBS containing 0.1% triton x-100. Just use a mounting medium with DAPI, unless you have other options. If you have confocal microscope and if you obtain images with 100X objective with 5x optical zoom at 0.3 micron intervals you should clearly see the nucleus and the rest of the cytoplasm and the plasma membrane. Usually, the labelling intensity should differ between the cytoplasm+plasma membrane compared to nucleus. Often you can also differentiate nucleolus as well.

Best wishes, Refik

I don't think you need to do so....you can just process at a time... you can use antibody against cadherin and antibody against your target together but different secondary antibodies-different source and color....mount with DAPI for nuclear staining....cadhherin will mark membrane........after merging the images obtained from different channels...you can be confirmed about localization............................to start off, you can simply merge with dapi image to see nuclear localization and the rest will appear in the edges of the cell

in my experience you do not really need to multiple and separate steps and incubations. Just do a regular IHC with PBS containing 0.1% triton x-100. Just use a mounting medium with DAPI, unless you have other options. If you have confocal microscope and if you obtain images with 100X objective with 5x optical zoom at 0.3 micron intervals you should clearly see the nucleus and the rest of the cytoplasm and the plasma membrane. Usually, the labelling intensity should differ between the cytoplasm+plasma membrane compared to nucleus. Often you can also differentiate nucleolus as well.

Best wishes, Refik

I agree with Reflk, in regards to the staining protocol, you should be able to do regular ICC, along with either a nuclei and/or membrane stain (depends on your research question) 

However, depending on the type of microscopy (epifluorescent or confocal) and optical components will determine what your pixel or voxel size needs to be to determine if signal from your two channels are related or not.

I would strongly suggest the use of a confocal microscope, with an oil immersion objective (can be 40x, 62x or 100x requires a numerical aperture > 1.4). To determine the sampling rate (voxel size) use the following website https://svi.nl/NyquistCalculator 

Once you have your images refer to http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1993886/ for performing analysis

If you want to follow the previously mentioned protocols you need to fix the cells first. Concerning phosphokinase receptors I have only experience with Trk receptors, but generally, after fixation (especially with aldehyde based fixatives) it is more difficult to immuno-label a protein when it is located to the cell membrane than when it is located intracellularly. You surely have checked the antibody's datasheet already and if there is no information regarding fixation you have to try. Generally cold (stored in freezer) methanol or acetone works better than for example formaldehyde for membrane bound proteins. If no fixation works I think it is a good idea you have to first stain unfixed cells (the epitope should be extracellular), then fix cells followed by labelling the intracellular proteins. The signal of the first staining should remain after fixation, but if it does not, try another fluorophore. Sorry if I have misinterpreted your question. Maybe you want to know how to distinguish the membrane and the nucleus located proteins, and not technically how you should do the staining.

I have used immunofluorescence to detect molecules where the location of the protein (nuclear, membrane or both) indicated which translocations were involved in a disease process. I found fixing cells for 1 minute in acetone / methanol 1:1 permeabilised the cells and gave decent morphology. Only one round of staining is necessary. I was able to see the distribution and photograph it successfully on a fluorescence microscope. Confocal microscopes also worked but weren't essential. Using DAPI for nuclear staining was useful. 

It would be good to know if your antibody recognizes the extra or intracellular domains of the receptor. You can always compare side by side samples stained with the same antibody: one that you permabilize and one that you do not permeabilize. This will work best if your antibody recognizes an extracellular domain of your receptor. I would also be careful of the different fixation methods, PFA will maintain membrane integrity more than acetone and/or methanol. If you can spare the samples and antibody, you can do a side-by-side of different fixation methods to see what gives you the best outcome.

Thanks everyone for the helpful replies!