I’m using coloc2 for the first time and I’m a bit confused RE: thresholding. I am analysing z stacks of 2 proteins that we see do not particularly colocalise (they tend to localise just because they are so spread out rather than being specifically localised). As they are not individual events in the cell - both proteins are spread out - one primarily in the nucleolus, the other in the cytoplasm - there is no specific ROI.
I import the images, split the channels to get red/green and create maximum intensity projections (my images are deconvolved). However, since I have no specific ROI as the proteins are diffuse, how do I deal with background and in what way should I be thresholding? I have read up on the colocalisation threshold but am unsure how to implement this in the actual analysis. Similarly, I am aware of background subtraction but am still unsure which is best to use. I am guessing I need to deal with the background but also know Pearson’s (which I am seeking to use) does not normally require thresholding.