A fixative suitable for in situ hybridization histochemistry.

We compared the morphology and stability of hybridization signals between paraffin sections of rat retina fixed with commonly used 4% paraformaldehyde/PBS and those fixed with a fixative containing glutaraldehyde in in situ hybridization histochemistry, using a digoxigenin-labeled RNA probe complementary for beta-galactoside alpha 2,6-sialyltransferase mRNA. Retinal detachment was frequently observed in the sections fixed with 4% paraformaldehyde-PBS, whereas the morphology was satisfactorily preserved in those fixed with either 0.5% glutaraldehyde, 4% paraformaldehyde-PBS, or 2.5% glutaraldehyde-PBS. Without glutaraldehyde, it was difficult to determine the most appropriate length of proteinase 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 glutaraldehyde to paraformaldehyde or with glutaraldehyde alone, it was easy to establish the appropriate time for the unmasking procedure, since intense mRNA signals were constant throughout the retina by proteinase K digestion for more than 30-40 min. Using a fixative that causes stronger cross-linking (e.g., glutaraldehyde) is recommended to improve not only the morphology but also the stability of hybridization signals in in situ hybridization histochemistry with paraffin embedding and digoxigenin-labeled RNA probes.

Two-dimensional gel electrophoretic analysis of lectin receptors in the bovine interphotoreceptor matrix.

Proteins and glycoproteins of the bovine interphotoreceptor matrix (IPM) with or without neuraminidase treatment was analysed by two-dimensional gel electrophoresis combined with Western blotting and staining with seven horseradish peroxidase-labeled lectins. More than 80 spots of proteins and glycoproteins were revealed on the gel. Nineteen spots (or groups of spots) were revealed by staining with five lectins [concanavalin A, wheat germ agglutinin (WGA), peanut agglutinin (PNA), Ricinus communis agglutinin-1 (RCA-1) and soybean agglutinin (SBA)]; some of those spots were specific for one lectin and others reacted with several lectins. We could not detect distinct spots reacting with Dolichos biflorus agglutinin or Ulex europaeus agglutinin-1. Neuraminidase digestions of the IPM increased and unmasked the binding spots for PNA, RCA-1 and SBA. The spots of WGA-receptors without neuraminidase treatment were mostly identical to the receptors for PNA, RCA-1 and SBA, which became prominent after the digestion. Spots reacting with RCA-1 were mostly identical to the spots of SBA-receptors. The spots reacting with PNA coincided only partially with the spots reacting with RCA-1 and SBA.