[Histonet] Re: YFP staining

[Johnson, Teri]

Hi James,

Have you checked under the microscope at your cytospins to verify there is YFP in the sample? That would be the first thing I looked at. Next, are you positive the anti-GFP antibody is cross-reacting with the YFP in your tissue samples? Do you have any non-YFP tissue samples that you have stained with the antibody? If you're sure the antibody is working properly, and there is YFP in your sample, then you should look at your fixation.

I think you're overfixing the cells. A cell monolayer doesn't require very long fixation. There's no connective tissue and layers upon layers of cells to penetrate and fix. I would cut that down to 1 hour, or even better do a time course using whatever timeline you think is reasonable and see if fixation plays a role in your staining.

Start there and see if that helps.

Best wishes,
Teri Johnson, HT(ASCP)QIHC
Managing Director, Histology Facility
Stowers Institute for Medical Research
Kansas City, MO