GFP cells in paraffin.....orange? - (Jul/05/2012 )
I have been cutting paraffin (10um thick) embedded murine myocardial tissue that has had GFP cells injected into non-GFP mycardium. I cut 1 slide and check the paraffin section under fluorescence only to notice 2 things.
1. On some slides with that tissue a potion of the outer edge (1-2 cells deep for about 1/5 of the outer area) will light up orange under a triple filter, green under fitc filter and red for a tritc filter but nothing under the Dapi filter. This is looking at the slide while the tissue is still paraffin bound. On a section further into the tissue this lit-up section will no longer be lit up, then cut a bit further and it will be lit up again. I know these are not the implanted GFP cells, but what would cause this?
2. When I have found cells that are believed to be GFP implanted, they light up under the triple filter, fitc filter and Tritc filter, but not under the DAPI filter. This is while embedded in paraffin on the slide.
When I stain them with GFP and DAPI, those injected cells do light up Green under the FITC filter and the nuclei light up blue under the DAPI filter...however, they also light up red under the TRITC filter eventhough no stain or secondary containing TRITC was used. What does this mean of the implanted cells that were stained for?
Some of what you are seeing is called autofluorescence - especially the ones where you see it on all filters. The edge effect is probably paraffin that has permeated the tissue and is fluorescing. You could dewax and check again.