I have been experiencing linearization in my on my dual staining plot, even after compensation. Linearization is also observed in neg ctrl (with 2' antibody), but I understd it is common to see that in neg ctrl. How do I overcome this issue?

Cell: Primary mucosal - consist of other cells type
1' Antibody: p75 & Thy1
2' Antibody: AF 488 & PE (direct conjugated with Thy1)

I have tried with and w/out PFA, reducing PFA concentration, titrated 2' antibody concentration (found 1:200 optimal). I still hv the same problem.

Do it go to do inherently with the cell type and the expression of this surface marker? In immunocytochemistry, it has very low expression (low fluor signals)

it is a worrying factor because p75 population is a very low % (8-15%) and slight linearization (up to 4%) can shift my results. I am trying to get a statistically significant analysis of these %, but my std deviation is so high.

What other factors can contribute to the linearization and how can I overcome them?