A FIXATIVE SUITABLE FOR INSITU HYBRIDIZATION HISTOCHEMISTRY

We compared the morphology and stability of hybridization signals betw een paraffin sections of rat retina fixed with commonly used 4% parafo rmaldehyde/PBS and those fixed with a fixative containing glutaraldehy de in in situ hybridization histochemistry, using a digoxigenin-labele d RNA probe complementary for beta-galactoside alpha2,6-sialyltransfer ase mRNA. Retinal detachment was frequently observed in the sections f ixed with 4% paraformaldehyde-PBS, whereas the morphology was satisfac torily preserved in those fixed with either 0.5% glutaraldehyde, 4% pa raformaldehyde-PBS, or 2.5% glutaraldehyde-PBS. Without glutaraldehyde , it was difficult to determine the most appropriate length of protein ase K digestion of tissue sections for facilitating probe penetration, since the optimal time for definite hybridization was variable among the retinal cells in heterogeneous layers. By addition of glutaraldehy de to paraformaldehyde or with glutaraldehyde alone, it was easy to es tablish the appropriate time for the unmasking procedure, since intens e mRNA signals were constant throughout the retina by proteinase K dig estion for more than 30-40 min. Using a fixative that causes stronger cross-linking (e.g., glutaraldehyde) is recommended to improve not onl y the morphology but also the stability of hybridization signals in in situ hybridization histochemistry with paraffin embedding and digoxig enin-labeled RNA probes.