Hello, I need to detect mCherry+ cells in vitro using fluorescence microscope. I am sure mCherry+ cells are there, but mCherry fluorescence may be decreased. I have tried conjugated mCherry-PercP (from Novus), Ds-Red and Ds-Red1 (both from Santa Cruz). All of them didn't work. I am confused, should Ds-Red antibody detect mCherry protein? What other antibodies can you recommend? Thanks in advance!
mCherry give a decent fluorescence in living and fixed cells. I never had problems with it. Are you processing the cells? If you used an antibody, are you doing intracellular staining? I need more information to understand your problem better. Try the following first.
1. check whether the mCherry is expressed (use flow cytometry to detect a shift in the peak)
2. check whether you are using right lasers and detection.
I hope it helps. Goodluck
Hello,
I fix the cells with 2%PFA, then permeabilize with Acetone:Methanol (50:50) or with Permeabilization buffer (Triton x100). Then I stain with antibody o/n.
1. Yes, I can detect mCherry by flow cytometry, but I need an in-vitro image. mCherry seems to be weak after the treatment I do ( I can see that by flow data), so I need to enhance it with a proper antibody in order to see under the microscope. Do you think Ds-Red antibody (Santa Cruz) should detect mCherry? It doesn't say on their infosheets
I have done fluorescence confocal microscopy and normal fluorescence microscopy with my cells. I just fixed the cells with 3% PFA for 20 min, then washing, followed by incubation with DAPI for 20 min and washing. I never had problems in detection. The mcherry expression in your cells might be low. or Check whether your microscope can detect mCherry or Texas RED.
Your protocol looks fine. I hope you are using 0.1% Triton x100. The antibody against DS-RED or RFP should work flawlessly. Other antibodies are Abcam (ab125096), antiRFP (ABIN129578).
Goodluck
Hi Olga,
I often use the 5F8 antibody from Chromotek:
https://www.chromotek.com/products/conventional-antibodies/rfp-antibody-5f8
Works excellent with mCherry and is the most highly cited RFP/mCherry antibody on citeAB:
https://www.citeab.com/search?q=rfp
Good luck with the experiment!
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