PLP-AMeX Method, Fixation Using PLP Fixative and Embedding in Paraffin by the AMeX Method, is Useful not only for Histochemistry but also In Situ Hybridization

Abstract

The authors have previously reported that the combination of fixation with periodate-lysine-paraformaldehyde (PLP) fixative and embedding in paraffin by the AMeX method (the PLP-AMeX method) resulted in better preservation of immunoreactivities and endogenous enzyme activities. In the present study, we investigated whether PLP-AMeX-processed tissue would be useful for in situ hybridization (ISH). The pancreases were removed from rats treated with or without streptozotocin (STZ), and was fixed in PLP fixative. After fixation, the specimens were processed and embedded in paraffin by the AMeX method. Paraffin sections were made on which ISH and immunohistochemical staining were performed. ISH was performed according to the non-radioactive method using digoxigenin-labeled oligodeoxynucleotides probes and anti-digoxigenin antibody. For ISH, a 28S rRNA antisense probe was used for evaluation of the level of hybridizable RNA in the sections. 28S rRNA was clearly detected in the various cells of the pancreas. Using an insulin antisense probe, the presence of insulin mRNA was clearly identified in the pancreatic islet-cells. Furthermore, optimal results for ISH of insulin mRNA and immunohistochemical staining of insulin could be obtained in consecutive sections from a PLP-AMeX-processed block. In the rats treated with STZ, the histological changes of pancreatic islet-cells and the changes in staining intensity of immunohistochemical staining for insulin protein and ISH for mRNA could be observed in the PLP-AMeX-processed sections. These findings suggest that hybridizable RNAs are well preserved by the PLP-AMeX method, and that the method is useful for ISH as well as immunohistochemical and enzyme histochemical analysis.