Hello all, 
This list has been very helpful to new people in the field. I work in
Orthopaedic surgery and I am having trouble with immunofluorescent staining bone
sections. It's not so much the autofluorescence; I avoid the GFP channel at all
costs and can distinguish real signal from background in the Green and IR
channels pretty well. The problem I recently have been running into is no DAPI
staining in my sections. 

These sections are heart valves removed from older people with end stage
calcified heart valves. The dapi staining is really weak or not even there, all
I see is lots of background in the UV filter. I can see where cells probably
are, based on the round shape and fluorescent signal in other channels, but it's
indistinguishable from non-cellular parts. I know the DAPI I am using works, I
have used it successfully in younger bone specimens. I checked the sample under
a confocal microscope, and it seems pretty clear that the DAPI is not staining
the nuclei, instead of it just staining everything. 

I recently learned that a colleague in the lab here is having similar problems
with his old bone specimens. A recently collected specimen which was quickly
fixed, processed, and embedded in parrafin is giving the same poor DAPI
staining. This is in stark contrast to bone specimens he has collected from
younger individuals, where the DAPI stains quite well. We can clearly see cells
in H&E/Saffranin-O stains, but the cells don't pickup any DAPI stain.

In both cases, the samples are formalin fixed and embedded with either parrafin
or paraplast, and then cut at 7 microns.  

Does anyone know why DAPI would not work in older tissue samples? Has anyone
else experienced this? 


Thank you very much, 
Kevin

-- 
Kevin Egan
Research Specialist
University of Pennsylvania
School of Medicine

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