Hi,
I use a EYFP cre reporter mice and do IHC on them to see the expression pattern. Usually, I slice the brain and on the same day start the IHC protocol with blocking and primary antibody incubation. After overnight incubation I do rest of the protocol; washing, secondary staining etc. and mount them on a slide. However, for the last IHC I did it a little different and stored the slices in a potassium based buffer saline with EDTA for 48 hours before starting the IHC procedure. I am not able to see any EYFP signal when excited with 488 laser and this was not the case before. This makes me think of two possible explanations: 1. somehow the animal got switched and wrong mice was put in the cage or 2. Nascent EYFP is lost due to 48 hour long storage before starting IHC.
I am more inclined to think it is the latter but I don't have much experience in doing slice IHC so I don't completely believe my hunches. Will appreciate any suggestion or troubleshooting from someone who have had similar experience.
Thanks!
Jimmy
Hi,
Storing these slices for 2 days in KPBS shouldn't affect the native eYFP signal, although I don't know if EDTA has an effect on that. If you need to store free-floating brain sections, you should try collecting them in a cryoprotectant solution and storing them at -20°C.
Recipe for cryoprotectant solution:
- Prepare buffer A: NaH2PO4, H2O at 27,6g/L (0.2M)
- Prepare buffer B: Na2HPO4, 2H2O at 35.6g/L (0.2M)
- Mix: 27.74mL buffer A + 96mL buffer B + 375mL H2O (ultra-pure) + 300mL ethylene glycol + 200mL glycerol
With the slices stored at -20°C in this solution, I usually keep the signal for YFP, Zsgreen and mCHerry for months (even years). If it doesn't work better, you should do an IHC against eYFP (any antibody against GFP should work).
Good luck!
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