Is there any chance you could give me some more information about the RFP protein you are using? I mean, I do work with calcium imaging using RFP proteins, and after the recordings, and never lost any fluorescence. So, I fix the cells (PFA 4% 10 min) and then I permeabilize the cells only with Triton X-100 at 0,05% for 45 min. Maybe you are being too aggressive using all these detergents during permeabilization?

Hope it helps!

Hi, I agree with Jùlia about your permeabilization protocol. If you don't solve the problem with a less aggressive permeabilization you can try to use a primary antibody anti-RFP, to amplificate the signal.

Hello Bernadette, if you see your signal after fixation, but not after permeabilization, it would suggest that you are losing your signal through the holes you are making. I presume your RFP-tagged molecules is small and soluble?

In that case, I would guess your problem is that you did not fix enough. For us (using GFP, not RFP though) fixing with 2% FA solved the issue. This protocol was adopted from Heinen et al. Cytometry 2014 (PMID: 24616430).

I hope this helps. Good luck, Gerhard

Thank you for the suggestions! Just to clarify, I didn't use all of those detergents at the same time - I tried them in separate combinations with one fixation method at a time (eg. 4% PFA with saponin, 4% PFA with Triton, 2% PFA with saponin, etc).

Even using 4% PFA for 30 minutes did not resolve the issue so I'm not sure if fixation is the problem here. The RFP fusion I'm using is localized to lipid membranes so that might be why I'm losing my signal after permeabilization.

For anti-RFP antibody, do you use that after permeabilization or incubate with the perm buffer of choice?

Dear Bernadette Anne Chua I suggested to use anti-RFP antibody as a primary antibody, so fixation - permeabilization - incubation with primary antibody - wash - secondary antibody. In my experience (not for FACS, but for IF), I used to amplificate the GFP signal with an anti-GFP.

However, permeabilization is really necessary? You can run an experiment by plating your cells, fix and permeabilize/not permeabilize.

Thank you, will try to see if that helps. I want to co-stain with proliferation markers so I would need to permeabilize.

Any idea why this would be affecting RFP primarily but not GFP?

For anyone reading in 2023, there is a solution coming soon from the startup I work at (LASE Innovation). We are commercializing instrumentation and Laser Particles for single-cell barcoding that enable you to acquire repeated flow measurements of the same cells. This lets you measure your samples' fluorescent signals at multiple points throughout a harsh processing protocol (before and after a harsh perm) at the points in time where signals are at their maximum intensities. The Laser Particles act as unique barcodes for the cells, and so your data is stitched together between measurements for every single cell. You end up with one FCS file that appears as though you measured everything at the same time. We recently showed at ISAC's CYTO conference in Montreal this recovers original GFP fluorescence through the harshest fix/perms (FOXP3 kit) in a case of intracellular cell cycle staining, outperforming anti-GFP staining. We also demonstrated the full recovery of fluorescence due to damaged antigen epitopes and degraded fluorochromes through a methanol perm in a phospho-flow protocol. If you are interested in becoming an early adopter please get in touch :-)