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

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