Combined Detection of Newly Synthesized RNA and Nuclear Proteins at the Ultrastructural Level: a Modification of the Protocol for Immunoelectron Microscopy

Resumo

In the present work, we propose a novel variant of a protocol that allows combined revealing of nascent RNA transcripts and representative proteins at the ultrastructural level using immunoelectron microscopy. Early mouse embryos injected with BrUTP were used as a test system. The standard procedure for processing of ultrathin sections with a mixture of the appropriate primary antibodies in this case gives unsatisfactory results, perhaps due to a mismatch of blocking buffers and diluents. We used a new variant of the technique, which contains two steps: a complete cycle of processing the sections on grids with an antibody to specific nuclear protein and a complete cycle of processing the same sections with an antibody that recognizes BrUTP. In the interval between the steps, the sections are allowed to dry, thereby eliminating the mixing of the buffer solutions used in the first and second cycles of immunolabeling. Here, we demonstrate the efficiency of this protocol and the specificity of the observed immunolabeling using examples of simultaneous detection of nascent RNA, as well as ATRX protein and a functional histone modification, H4K5ас.