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Biology

In Situ Exploration of Murine Megakaryopoiesis using Transmission Electron Microscopy

Published: September 8th, 2021

DOI:

10.3791/62494

1Université de Strasbourg, INSERM, EFS Grand Est, BPPS UMR-S 1255, FMTS

Here, we present a protocol to analyze ultrastructure of the megakaryocytes in situ using transmission electron microscopy (TEM). Murine bone marrows are collected, fixed, embedded in epoxy resin and cut in ultrathin sections. After contrast staining, the bone marrow is observed under a TEM microscope at 120 kV.

Differentiation and maturation of megakaryocytes occur in close association with the cellular and extracellular components of the bone marrow. These processes are characterized by the gradual appearance of essential structures in the megakaryocyte cytoplasm such as a polyploid and polylobulated nucleus, an internal membrane network called demarcation membrane system (DMS) and the dense and alpha granules that will be found in circulating platelets. In this article, we describe a standardized protocol for the in situ ultrastructural study of murine megakaryocytes using transmission electron microscopy (TEM), allowing for the identification of key characteristics defining their maturation stage and cellular density in the bone marrow. Bone marrows are flushed, fixed, dehydrated in ethanol, embedded in plastic resin, and mounted for generating cross-sections. Semi-thin and thin sections are prepared for histological and TEM observations, respectively. This method can be used for any bone marrow cell, in any EM facility and has the advantage of using small sample sizes allowing for the combination of several imaging approaches on the same mouse.

Megakaryocytes are specialized large polyploid cells, localized in the bone marrow, responsible for platelet production1. They originate from hematopoietic stem cells through an intricate maturation process, during which megakaryocyte precursors progressively increase in size, while undergoing extensive concomitant morphologic changes in the cytoplasm and nucleus2. During maturation, megakaryocytes develop a number of distinguishable structural elements including: a polylobulated nucleus, invaginations of the surface membrane that form the demarcation membrane system (DMS), a peripheral zone devoid of organelles surround....

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All animal experiments were performed in accordance with European standards 2010/63/EU and the CREMEAS Committee on the Ethics of Animal Experiments of the University of Strasbourg (Comité Régional d'Ethique en Matière d'Expérimentation Animale Strasbourg). The protocol is schematically shown in Figure 1.

1. Bone marrow collection and fixation ( Figure 1A)

CAUTION: This proced.......

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Bone marrow histology
Observation of the bone marrow toluidine blue histology under a light microscope is key to rapidly analyze the overall tissue architecture in terms of e.g., tissue compactness, microvessel continuity, and the size and shape of megakaryocytes (Figure 1D). It is performed before ultrathin sections to determine the need of cutting deeper in the bone marrow block. Due to their giant size and nuclear lobulation, the more mature megakar.......

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Direct examination of megakaryocytes in their native environment is essential to understand megakaryopoiesis and platelet formation. In this manuscript, we provide a transmission electron microscopy method combining bone marrow flushing and fixation by immersion, allowing to dissect in situ the morphology characteristics of the entire process of megakaryocyte morphogenesis taking place in the bone marrow.

The flushing of the bone marrow is a critical step of this method, as the succes.......

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The authors wish to thank Fabienne Proamer, Jean-Yves Rinckel, David Hoffmann, Monique Freund for technical assistance. This work was supported by ARMESA (Association de Recherche et Développement en Médecine et Santé Publique), the European Union through the European Regional Development Fund (ERDF) and by Grant ANR-17-CE14-0001-01 to H.d.S.

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Name Company Catalog Number Comments
2,4,6-Tri(dimethylaminomethyl)phenol (DMP-30) Ladd Research Industries, USA 21310
Agarose type LM-3 Low Melting Point Agar Electron Microscopy Sciences, USA 1670-B
CaCl2 Calcium chloride hexahydrate Merck, Germany 2083
Copper grids 200 mesh thin-bar Oxford Instrument, Agar Scientifics, England T200-CU
Dimethylarsinic acid sodium salt trihydrate Merck, Germany 8.20670.0250
Dodecenyl Succinic Anhydride (DDSA) Ladd Research Industries, USA 21340
Double Edge Stainless Razor blade Electron Microscopy Sciences-EMS, USA EM-72000
Ethanol absolut VWR International, France 20821296
Filter paper, 90 mm diameter Whatman, England 512-0326
Flat embedding silicone mould Oxford Instrument, Agar Scientific, England G3533
Glutaraldehyde 25% Electron Microscopy Sciences-EMS, USA 16210
Heat plate Leica EMMP Leica Microsystems GmbH, Austria 705402
Histo Diamond Knife 45° Diatome, Switzerland 1044797
JEOL 2100 Plus TEM microscope JEOL, Japan EM-21001BU
Lead citrate - Ultrostain 2 Leica Microsystems GmbH, Austria 70 55 30 22
LX-112 resin Ladd Research Industries, USA 21310
MgCl2 Magnesium chloride hexahydrate Sigma, France M2393-100g
Mounting medium - Poly(butyl methacrylate-co-methyl methacrylate) Electron Microscopy Sciences-EMS, USA 15320
Nadic Methyl Anhydride (NMA) Ladd Research Industries, USA 21350
Osmium tetroxide 2% Merck, Germany 19172
Propylene oxide (1.2-epoxypropane) Sigma, France 82320-250ML
Saline injectable solution 0.9% NaCl C.D.M Lavoisier, France MA 575 420 6
Scalpel Surgical steel blade Swann-Morton, England .0508
Sodium tetraborate - Borax Sigma, France B-9876
Sucrose Merck, Germany 84100-1KG
Syringe filter 0.2µm Pall Corporation, USA 514-4126
Toluidine blue Ladd Research Industries, USA N10-70975
Trimmer EM TRIM2 Leica Microsystems GmbH, Austria 702801
Ultramicrotome Ultracut UCT Leica Microsystems GmbH, Austria 656201
Uranyl acetate Ladd Research Industries, USA 23620

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