Transmission Electron Microscopy to Quantify Glycogen Distribution in Human Skeletal Muscles

Abstract

Source: Jensen, R., et al. Quantification of Subcellular Glycogen Distribution in Skeletal Muscle Fibers using Transmission Electron Microscopy. J. Vis. Exp. (2022)

In this video, we demonstrate the sample preparation of human skeletal muscle tissue and its staining for transmission electron microscopy to visualize and quantify subcellular glycogen distribution.

Protocol

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board 1. Primary fixation, post-fixation, embedding, sectioning, and contrasting Prepare 1.6 mL of primary fixative solution (2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.3)) in a 2 mL micro centrifugation tube. Store it at 5 °C for …

Representative Results

Figure 1: Imaging and fiber typing. (A) Each fiber is imaged in a randomized systematic order. (B) Example of an image from the subsarcolemmal space. (C) Example of an image from the myofibrillar space. (D) In each myofibrillar image, the width of one Z-disc is measured (red lines). The measurements of a total of 12 Z-discs (one per image) give …

Materials

1,2-Propylene oxide	Merck	75-56-9
Embedding 812 resin medium kit	Taab	T031
Glutaraldehyde solution 25%	Merck	1.04239.0250
ITEM	Olympus		Imaging software
Leica EM AC20	Leica		Automatic contrasting system
OSIS Veleta digital camera	Olympus
Osmium tetroxide 4% solution	Polysciences	0972A
Philips CM 100 Transmission EM	Philips
Potassium hexacyanoferrate (II) trihydrate	Sigma-Aldrich	455989-245G
Sodium cacodylatbuffer 0.2 M ph 7.4	Ampliqon.com	AMPQ40989.0500
Ultra-microtome Leica UC7	Leica
Ultrostain lead citrate 3%, stabilized solution	Leica	16707235
Uranyl acetate dihydrate	Polysciences	6159-44-0