A NEW MONOCLONAL-ANTIBODY FOR SPECIFIC IMMUNOCYTOCHEMICAL STAINING OFNUCLEOLI

We have isolated a hybridoma cell line (clone 1E10) producing a monocl onal antibody which specifically recognizes nucleoli. The antibody (Ig M, k isotype) was found to react in a nucleolar pattern with a variety of cell types. Specific staining was only obtained on cryostat sectio ns of unfixed tissues. Paraffin embedding destroyed the epitope. Tissu e specificity or species specificity was not observed. Nucleoli in neo plastic cells were highly reactive, presumably due to the larger size of nucleoli in these cells. Immunoelectronmicroscopy (using a pre-embe dding as well as a post-embedding technique) confirmed the specific nu cleolar localization of the immunoreactivity. Immunoreactivity was con fined to the granular component of the nucleolus. The intensity of the immunoreactivity increased after cell or tissue pretreatment with DNa se, pronase or trypsin, indicating that the target epitope is not DNA or a protein. On Western blots of immunoreactive cells no specific sig nal was obtained, which supports the non-protein nature of the epitope . Acid hydrolysis and RNase digestion abolished the immunoreactivity. Parallel staining experiments with methylgreen pyronin and acridin ora nge confirmed the RNA nature of the epitope. In spot blots, immunoreac tivity was not found with tRNA or mRNA. These observations indicate th at 1E10 recognizes a conformational RNA epitope which occurs only in t he nucleolus.