Development of a fluorescence contrasting immunostaining technique for visualizing 3D astrocytic ultramorphology

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Abstract

Changes in astrocytic ultramorphology may underlie the development of neurodegenerative processes in their early stages. However, the mechanisms of its change are still poorly understood, since the size of the peripheral astrocytic processes forming the basis of the astrocytic synaptic coating are beyond the resolution of most optical microscopy (OM) methods. In turn, the disadvantage of promising methods of electron and scanning probe microscopy (EM and SPM) for such studies is the inability to determine the target area of the study due to the simultaneous use of fluorescence microscopy of immunocolored cells and the possibility of full-fledged 3D analysis of samples. In this paper, we consider the concept of solving the above problem by using an instrumental approach that combines the methods of SPM and OM together with ultramicrotomy as a method of restoring the 3D structure of the sample within a single hardware complex. To implement the proposed combined technique (optical-probe nanotomography, OPNT), the first stage of creating specialized fluorescent-contrasting labels based on conjugates of fluorescent semiconductor nanocrystals and single-domain antibodies has been developed in this work. This type of label will provide both immuno-staining of the “area of interest" for the restoration of 3D astrocytic ultramorphology, and contrast of astrocytes by the SPM method.

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About the authors

К. Е. Mochalov

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry

Email: voleinik@mail.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997

О. I. Sutyagina

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry; Koltzov Institute of Developmental Biology of Russian Academy of Sciences

Email: voleinik@mail.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997; ul. Vavilova 26, Moscow, 119334

А. V. Altunina

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry; Moscow Institute of Physics and Technology (National Research University)

Email: voleinik@mail.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997; Institutskiy per. 9, Dolgoprudny, 141701

D. О. Solovieva

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry

Email: voleinik@mail.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997

А. Е. Efimov

V.I. Shumakov Federal Research Center of Transplantology and Artificial Organs

Email: voleinik@mail.ru

Laboratory of Bionanotechology

Russian Federation, ul. Shchukinskaya 1, Moscow, 123182

V. A. Zhuchkov

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry

Email: voleinik@mail.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997

S. P. Chumakov

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry

Email: voleinik@mail.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997

V. A. Oleinikov

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry; National Research Nuclear University “MEPhI” (Moscow Engineering Physics Institute)

Author for correspondence.
Email: voleinik@mail.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997; Kashirskoe shosse 31, Moscow, 115409

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2. Fig. 1. The concept of using fluorescent-contrasting labels based on conjugates of nanoantibodies (NT) and fluorescent semiconductor nanocrystals (NC) for visualization of the volumetric ultrastructure of astrocytes using the OZNT method.

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3. Fig. 2. Visualization of astrocytes in the hippocampus of C57Bl6 mice (stratum radiatyum zone). (a) Immunocytochemical staining using IgG-GFAP antibodies; (b) immunocytochemical staining using E9-GFAP nanoantibodies; (c) astrocyte density in the stratum radiatum zone (number of astrocytes per 250,000 μm2) with IgG-GFAP and E9-GFAP staining; (d) 2D Sholl analysis profile (number of intersections of astrocytic processes with concentric spheres centered in the middle of the cell soma) of single astrocytes with IgG-GFAP and E9-GFAP staining.

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