Leica Microsystems EM AFS2 Application Note

Brand: Leica Microsystems

Model: EM AFS2

Category: Sandwich makers

Type: Application Note

This manual is also suitable for

EM ICE

Application Note

Structural study of

Caenorhabditis elegans

related instruments Leica EM ICE, Leica EM AFS2

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Structural study of

Caenorhabditis elegans

MATERIALS AND METHODS

Wildtype L4 stage C. elegans (N2 strain) were placed in the 100 µm deep side of Lecithin-coated (see detailed protocol*) type A 3 mm

Cu/Au carriers (Leica) with extracellular ﬁller containing 1% (w/v) Agarose type IX and 2% (w/v) Bovine Serum Albumin in bacteria

medium (see preparation details**) and sandwiched with the ﬂat side of Lecithin-coated type B 3 mm Cu/Au carriers (Leica). Samples

were frozen in a high-pressure freezer (EM ICE, Leica). After freezing sandwiches were opened in liquid nitrogen and the type A carriers

were transferred into ﬂow-through capsules (plastic capsules D5 x H15 mm, Leica) to the automated freeze-substitution device (AFS2,

Leica) set at –90 °C. Flow-through capsules with samples were placed into 2.0 ml Sarstedt tubes containing 800 µl precooled 2% (w/v)

Osmium tetroxide in anhydrous Methanol (see modiﬁed “Karreman” protocol***). The rubber rings from the lids of these Sarstedt tubes

have been removed.

Samples were kept at –90 °C for 48 h in this solution, next washed 4x 30 min at –90 °C with 500 µl precooled 2% (w/v) Osmium

tetroxide in anhydrous Acetone and ﬁnally left for 46 hat –90 °C in 2.0 ml Sarstedt tubes containing 500 µl precooled 2% (w/v) Osmium

tetroxide in anhydrous Acetone. Washing steps were carried out by transferring the ﬂow-through capsules with samples to 2.0 ml

Sarstedt tubes containing 500 µl precooled 2% (w/v) Osmium tetroxide in anhydrous Acetone.

Next samples were gradually warmed from –90 °C to –60 °C (increment of 2 °C/h); kept at –60 °C for 8 h before warmed up to –30 °C

(increment of 2 °C/h). At –30 °C samples were kept for 8 h and ﬁnally were placed on ice for 1 h followed by 1 h at room temperature.

Samples were washed 3x 5 min with anhydrous Acetone by transferring the ﬂow-through capsules with samples to 1.5 ml Eppendorf

tubes containing 500 µl anhydrous Acetone. A volume of 800 µl freeze-substitution medium was chosen to have enough capacity to

replace the water in the sample for Acetone. For all next steps a volume of 500 µl was used to prevent loss of the sample out of the

ﬂow-through capsule by overﬂow.

After the last wash with anhydrous Acetone samples were inﬁltrated with Epon resin (see detailed protocol****). Inﬁltration with Epon

resin/ Acetone mixtures was perfor¬med at room temperature by the following steps:

Epon : Acetone = 1 : 2 for 2 h (33% (v/v) Epon)

Epon : Acetone = 1 : 1 for 18 h (50% (v/v) Epon)

Epon : Acetone = 2 : 1 for 8 h (66% (v/v) Epon)

Epon : Acetone = 3 : 1 for 18 h (75% (v/v) Epon)

pure Epon for 8 h (100% Epon)

pure Epon for 18 h (100% Epon)

pure Epon for 6 h (100% Epon)

Finally, individual C. elegans were taken out of the type A carriers and placed separately in the cavities of an embedding mould (Aclar

scientiﬁc). The cavities were ﬁlled with pure EPON and polymerized for 72 h at 65 °C. Approximately 70 nm thick sections were cut on

an ultramicrotome (Ultracut UCT, Leica) and collected on Carbon coated-Formvar-50 mesh hexagonal copper grids. The sections were

contrasted 6 min with 7% (w/v) Uranyl Acetate in 70% Methanol and 2 min with Reynolds Lead Citrate.

Micrographs were acquired on a Tecnai 12 electron microscope (FEI) operated at 100 kV and spotsize 3. Electron micrographs (Binning1,

2048x2048 pixels) were collected with a TIETZ camera (TIETZ TVIPS TemCam F214) at 15,000x magniﬁcation (pixel size 0.375nm).

Courtesy: Elly van Donselaar

, Martin Harterink

, Karin Vocking

and Rob Mesman

1 Universiteit Utrecht, The Netherlands, 2 Radboud Universiteit Nijmegen, The Netherlands

* Lecithin coating of carriers:

Prepare a solution of 2% (w/v) Lecithin (L-α-Phosphatidylcholine from egg yolk, Fluka-61755) in Chloroform. Dip the type A carriers one

by one in the Lecithin solution and lay the carriers with the 100 µm deep side up on ﬁlter paper. Dip also the type B carriers in the

Lecithin solution and lay the carriers with the ﬂat side up on ﬁlter paper. The carriers will be covered with a thin layer of Lecithin after

the Chloroform has evaporated.

RESULTS

Fig. 1

Caenorahbditis elegans

C. elegans

processed as described

was sectioned perpendicular just

behind the head at the position of the

kidneys. Overview of a section

reveals the subcellular organization

of cell of interest taken from the

lateral side of

C. elegans.

** C.elegans extracellular ﬁller:

For 2 ml: 20 mg Agarose, type IX (Ultra-low gellingpoint Agarose, Sigma-Aldrich A-5030) in 1.6 ml bacteria medium, dissolve at 60 °C.

Let the solution cool down to 37 °C, add 400 µl 10% (w/v) BSA (Bovine Serum Albumin, fraction V, Sigma-Aldrich A-9647) in milliQ water

and mix gently.

*** Procedure described in “One Protocol Fits All” Matthia Karreman et al.: Lights will guide you, Sample Preparation and Applications

of integrated Laser and Electron Microscopy, published by Uitgeverij BOXPress, ‘s-Hertogenbosch, The Netherlands, ISBN: 978-90-889-

1562-8

**** Epon resin:

For approximately 50 ml pure Epon add together:

1. 21.5 gram Epoxy embedding medium (Fluka)

2. 25.0 gram Araldite M Hardener 964 (Fluka)

3. 7.0 gram Epoxy embedding medium hardener MNA (Fluka)

Mix well for at least 10 min and add:

4. 0.75 ml Epoxy embedding medium accelerator DMP 30 (Fluka)

Mix well for at least another 10 min

LNT Application Note - CAENORHABDITIS ELEGANS

TEC

Fig. 2: Higher magnification of the selected area in Fig. 1

LD-Lipid Droplet, M-Mitochondria, ER-Endoplasmatic reticulum, G-Golgi

Fig. 3: Higher magnification of the selected area in Fig. 1

TEC-Tubular Excretory Cell, M-Mitochondria

Leica Microsystems EM AFS2 Application Note

Brand: Leica Microsystems

Model: EM AFS2

Category: Sandwich makers

Type: Application Note

This manual is also suitable for

EM ICE

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Leica Microsystems EM AFS2 Application Note

This manual is also suitable for

Leica Microsystems EM AFS2 Application Note

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