Microwave pretreatment improves RNA-ISH in various formalin-fixed tissues using a uniform protocol

Abstract

RNA is situ hybridization technique (RNA-ISH) is hampered by formalin fixation of tissues. This necessitates a pretreatment step of enzymatic digestion. However, to achieve the best results, digestion times and concentrations of enzymes have to be adapted for every specimen in a cumbersome procedure. Microwave pretreatment (MP) of formalin-fixed tissues has developed to become a powerful tool in immunohistochemistry (IHC) in recent years. To evaluate whether MP could also be helpful for RNA-ISH, we compared MP and conventional enzymatic pretreatment systematically using different tissues and various fixation times. As a model for RNA-ISH we chose the detection of kappa/lambda light chain mRNA in tonsils and lymph nodes with follicular hyperplasia and lymph nodes of patients with lymphocyte predominant Hodgkin's Disease (LPHD). Signal intensity obtained after MP was at least as good or dramatically enhanced as that obtained with optimized, single case adapted conventional pretreatment, the morphology being much better preserved after MP. These results confirm MP as a suitable method to unify the staining protocols in RNA-ISH, regardless of the duration of formalin fixation. Based on our results we recommend MP as a reliable and inexpensive method to enhance, standardized and simplify RNA-ISH.