Fri Sep 09, 2016 2:23 pm by AdeebR
Hi Kate,
As several others have already suggested, our typical preferred workflow for phospho signaling is to stimulate, then do a "mild" fix with 1% formaldehyde for 10 mins, surface stain, and then proceed to methanol permeabilization before staining for the intracellular phospho proteins. Using this protocol we typically have no issues resolving most major immune subsets (CD14+ expression using clone M5E2 is usually easy to resolve).
However, there certainly are some antibodies where do do lose staining even with mild fixation, and in these cases the only option we've found is to add the antibodies to the cells prior to fixation. While you observed that some antibodies can block stimulation, we've also come across situations in which antibodies can induce signaling, or modulate signaling pathways, which can confound your results. The effects can sometime be subtle, so whenever we take this approach we always do pilot experiments where we test our whole signaling panel and stimulation protocol with and without the addition of the staining antibodies to determine whether any of the pathways are being affected.
This can be pretty laborious and expensive, so we typically only go through with this only if determining expression of the fixation sensitive protein in direct conjunction with signaling profiling is absolutely critical for an experiment (in many experimental setups it isn't). If it's not, it's generally much easier to just run parallel aliquots of the samples (i.e., one where we include the surface markers on the cells prior to fixation, and another where we look at signaling), and include a sufficiently large panel of fixation resistant surface markers to be able to identify the analogous populations in both samples where you want to compare protein expression.
Adeeb
Adeeb Rahman
Icahn School of Medicine at Mount Sinai, NYC