Combined Detection of Newly Synthesized RNA and Nuclear Proteins at the Ultrastructural Level: a Modification of the Protocol for Immunoelectron Microscopy
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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.
Keywords:immunoelectron microscopy double labeling BrUTP mouse embryos
This work was performed according to state assignment no. 0124-2018-0003, CITS assurance no. AAAA-A17-117032350035-4.
COMPLIANCE WITH ETHICAL STANDARDS
Сonflict of interests. The authors declare that they have no conflict of interest.
Statement on the welfare of animals. The study was approved by the Animal Ethics Committee of the Institute of Cytology of the Russian Academy of Sciences (Assurance Identification number F18-00380).
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