ИСТИНА

The cytoskeleton of endothelial cells, similarly to cytoskeletons of other animal cells, is represented by three types of filaments, which interact on the structural, functional and regulatory levels: microtubules, actin filaments and intermediate filaments. The interaction of microtubules with actin filaments was found to be important for functional activities of endothelial cells, especially for their barrier function. Identification of possible links between microtubules and actin structures may help us to understand how these cytoskeletal components cooperation is organized in functional endothelium. Endothelial cells express two isoforms of non-muscle actins (β- and γ-) and form β- and γ-actin structures simply identified by immunostaining with specific antibodies on the light microscopy level, but structurally undistinguishable on electron microscopy (TEM) level. Optimal procedures of fixation and sample preparation are different for the visualization of microtubules and actin filaments on light microscopy and TEM: glutaraldehyde fixation is the most effective for TEM, but it is not quite appropriate for immunostaining of microtubules or β- and γ-actin structures. Therefore the aim of current work was experimental selection of the optimal fixation procedure for the visualization of cytoskeleton structures of endothelial cells for both light microscopy (confocal and super-resolution microscopy) and TEM. As a result, we proposed a complex fixation of endothelial cells for correlative super-resolution and TEM microscopy, which includes consecutive glutaraldehyde (2,5 %) and methanol (-20oC) fixation following by NaBH4 (0.2 %) treatment. This protocol allows to investigate fine colocalization of endothelial cell microtubules and actin structures formed by different isoforms (β- and γ-actins) via methods of double-immunostaining for confocal and super-resolution microscopy and for TEM. Supported by RFBR (#15-04- 08550) and MSU Development Program PNR 5.13.