[Histonet] Collagen-I gel, dye and autofluorescence

[Tim Demuth]

Hi there,

we are currently trying to cut FFPE sections of multicellular
tumor-spheroids grown in collagen-I gel (bovine). There are two major
problems:
1) Since the spheroid is so small (diameter ~200-500µm) and very pale it is
extremely hard to identify in the fixed and embedded collagen block. We
would like to use a dye to visualize the spheroid prior to embedding, so we
have some guidance as to where and when to expect the spheroid so we don't
have to cut through the whole block. Eosin works fine for that purpose,
however it's autofluorescence interferes with fluorescence IHC. Is there any
dye that doesn't show autof.?

2) We also experienced the spheroid to pop out of the gel when sectioning
resulting in an empty spot on the slide. Any suggestions on how to improve
fixation (currently 10% neutral buffered PFA for 24hrs) technique?

Thanks for the input, it's greatly appreciated!

Tim


Translational Genomics Research Institute, TGEN
Phoenix AZ