Periodic acid incubation can replace hydrochloric acid hydrolysis and trypsin digestion in immunogold--silver staining of bromodeoxyuridine incorporation in plastic sections and allows the PAS reaction.
We have examined the possibility of improving the present methods of detecting bromodeoxyuridine (BrdU) and for combining the PAS reaction with the BrdU detection by means of immunogold-silver staining (IGSS). This was done in testes fixed in Carnoy or Bouin, and in parts of the small intestine which were fixed in Carnoy or periodate-lysine-paraformaldehyde (PLP). All tissues were embedded in a mixture of glycol methacrylate and butanediol-monoacrylate. It was found to be impossible to carry out BrdU detection using HCl hydrolysis and trypsin digestion in combination with a PAS reaction. However, incubation of the plastic sections in periodic acid for a period of 30 minutes appeared to make it possible to eliminate the HCl denaturation step and to carry out a specific PAS reaction. Moreover, after incubation in periodic acid, trypsin digestion was no longer required to make the BrdU label accessible in GMA-embedded sections, nor to re-expose the antigenic sites in plastic sections of tissues fixed with cross-linking fixatives. In this way the loss of cell structures, which is inevitable when trypsin is used, can be avoided. Now a BrdU detection with improved morphology can be combined with the PAS reaction in the same plastic section in order to stain tissue carbohydrates. This is important for tumour diagnosis, where the PAS reaction can be very useful.
van de Kant
H J
HJ
Department of Cell Biology, State University of Utrecht, Medical School, The Netherlands.
de Rooij
D G
DG
D000818:Animals; D001973:Bromodeoxyuridine; D004247:DNA / Q000032:analysis; D016591:Histocytological Preparation Techniques; D006851:Hydrochloric Acid; D007150:Immunohistochemistry; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D010504:Periodic Acid; D051381:Rats; D016622:Silver Staining; D013737:Testis / Q000737:chemistry*; D014357:Trypsin
D010504:Periodic Acid; D004247:DNA; D014357:Trypsin; D001973:Bromodeoxyuridine; D006851:Hydrochloric Acid