Analysis of Lipid Droplets in Cardiac Muscle - Methods in Cell Biology - page 133

have been published and still do not offer quality resolution and information
( Kuramoto et al., 2012; Wang et al., 2013 ). Conventional TEM has been commonly
used to image cardiac LDs ( Chiu et al., 2001, 2005; Chokshi et al., 2012; Liu et al.,
2007; Pollak et al., 2013; Son et al., 2007; Wang et al., 2013; Zimmermann et al.,
2004 ) and revealed the close physical association between LDs and mitochondria.
We are describing here an optimized protocol to allow imaging of these organelles
and morphometry analyses of LDs and mitochondria.

8.2.1 Materials
8.2.1.1 Reagents and buffers
1. Mixture of 4% paraformaldehyde and 1% glutaraldehyde in phosphate buffer:
mix 2.5 ml 16% paraformaldehyde (Electron Microscopy Sciences, cat. no.
15710), 1 ml 10% glutaraldehyde (Electron Microscopy Sciences, cat. no.
16120), 5 ml 200 mM phosphate buffer and adjust final volume to 10 ml with
water.
2. Mixture of 2% paraformaldehyde and 2.5% glutaraldehyde in phosphate buffer:
mix 1.25 ml 16% paraformaldehyde (MP Biomedical, USA, cat. no.
0219998320), 2.5 ml 10% glutaraldehyde (MP Biomedical, USA, cat. no.
02189659510), 5 ml 200 mM phosphate buffer, and adjust final volume to 10 ml
with water.
3. Uranyl acetate: 3% solution in water, stored in the dark at 4 C.
4. Ethanol (C 2 H 5 OH): 30%, 50%, 70%, 90%, 100% solutions in water.
5. Spurr's resin (EMS): mix 8.0 g DER 736 (diglycidyl ether of propylene glycol),
10.0 g ERL 4221, 25.0 g NSA (nonenyl succinic anhydride), and 0.3 g DMAE
(dimethylaminoethanol), for a total volume of 40 ml.

6. Toluidine blue stain: mix one part deionized water, one part 5% (w/v) toluidine
blue, and one part 2% (w/v) sodium borate, filter, and store at room temperature.
Phosphate buffer: 200 mM solution, pH 6.8. Prepare solution A (200 mM
monobasic sodium phosphate) and solution B (200 mM dibasic sodium
phosphate). Mix 190 ml of solution A with 810 ml of solution B and adjust pH to
7.4 by using additional Solution A or B.

8.2.2 Equipment
1. Transmission electron microscope, 120 keV (Tecnai T12, FEI Co.)

8.2.3 Experimental design
1. Mice are euthanized by cervical dislocation before dissection. Left heart
ventricle tissue is excised quickly and soaked immediately in 4%
paraformaldehyde and 1% glutaraldehyde in phosphate buffer (Trump's
Fixative) and cut into roughly 5 mm 2 pieces using a razor blade and stored at
4 C in fixative.