MICROWAVE PRETREATMENT IMPROVES RNA-ISH IN VARIOUS FORMALIN-FIXED TISSUES USING A UNIFORM PROTOCOL

RNA in situ hybridization technique (RNA-ISH) is hampered by formalin fixation of tissues. This necessitates a pretreatment step of enzymati c digestion. However to achieve the best results, digestion times and concentrations of enzymes have to be adapted for every specimen in a c umbersome procedure. Microwave pretreatment (MP) of formalin-fixed tis sues 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 syste matically using different tissues and various fixation times. As a mod el for RNA-ISH we chose the detection of kappa/lambda light chain mRNA in tonsils and lymph nodes with follicular hyperplasia and lymph node s of patients with lymphocyte predominant Hodgkin's Disease (LPHD). Si gnal intensity obtained after MP was at least as good or dramatically enhanced as that obtained with optimized single case adapted conventio nal pretreatment, the morphology being much better preserved after MP. These results confirm MP as a suitable method to unify the staining p rotocols in RNA-ISH, regardless of the duration of formalin fixation. Based on our results we recommend MP as a reliable and inexpensive met hod to enhance, standardize and simplify RNA-ISH.