In Situ Exploration of Murine Megakaryopoiesis using Transmission Electron Microscopy

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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.......

Discussion

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.......

Materials

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