This is my first contribution to the Microforum and it deals with the following problem:
I am planning to perform multiplexed immunofluorescence on FFPE tissue sections in order to find out which cell types present in the tissue express particular proteins of interest. For this purpose I want to use CellProfiler to identify all the cells based on DAPI (primary object recognition) and define the cell boundaries/shape based on cell surface/morphology (e.g. membrane protein) staining (secondary object recognition). The latter should help me to define the “cell body” in order to actually determine which cells are “positive” for certain proteins (i.e. make CellProfiler search for positive staining within the secondary object masks).
Now I am facing the following challenges when looking for an appropriate way to stain the “cell bodies”:
- CellMask stains (Thermo Fisher) nicely stain the cell surface, but they are lipid-binding reagents that do not work on FFPE.
- Phalloidin-based staining (F-actin) is an option for FFPE, but I am worried that the cell borders are not defined with good enough contrast (especially in regions with high cell densities), which will make it very hard to define the cell borders (secondary masks) in CellProfiler (I might be wrong though, so please let me know if you had good experience with it…)
- A good option would be to stain cell membrane proteins, but since I do not want to stain separate membrane proteins for each possible cell type present in the tissue, I am looking for a very ubiquitous membrane protein, which is expressed on virtually every cell.
Now to the actual question: Is anyone aware of (or has anyone ever successfully used antibodies against) a very unspecific/ubiquitous membrane protein that could serve as a cell type-independent cell surface marker to determine cell shape? Or are there other alternatives to the above-mentioned that are compatible with FFPE?
Otherwise I would try phalloidin or, in the worst case, use CellProfiler’s “MeasureObjectNeighbors” module to define a specific radius around the nucleus as the “cell body”, which of course would be way more “dirty”.
I am looking forward to a good discussion with plenty of suggestions from more experienced people!