Leica Microsystems EM AFS2 Application Note

Application Note

Cultured Rat Hippocampal Neurons

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Cultured Rat Hippocampal Neurons

MATERIAL AND METHODS

Rat Hippocampal neurons, cultured on 50 µm thick Aclar (Aclar embedding ﬁlm, EMS) for 19 days, were frozen in the 100 µm deep side

of lecithin coated (detailed protocol Appendix I) type A 3 mm Cu/Au carriers (Leica) and sandwiched with the ﬂat side of lecithin coated

type B 3 mm Cu/Au carriers (Leica). No additional ﬁller was used, only cell culture medium with the addition of Hepes buffer pH 7.2 to

a ﬁnal concentration of 25 mM. 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 au-

tomated freeze-substitution device (AFS2, Leica) set at –90 °C. Flow-through capsules with samples were placed into Sarstedt tubes

with 800 µl Müller’s freeze-substitution medium (Müller et.al., 1980), containing 3% (v/v) Glutaraldehyde + 1% (w/v) Osmium tetroxide

and 0.3% (w/v) Uranyl Acetate in anhydrous Methanol (detailed protocol Appendix II).

Samples were kept at –90 °C for 48 h in this solution and next 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 warmed up from –30

°C to +2 ˚C (increment of 10 °C/h) and kept at this temperature for 2 h. After this last substitution step samples were washed 2x 5 min

with 500 µl anhydrous Methanol, 1x 5 min with 500 µl 50% anhydrous Methanol + 50% anhydrous Acetone and ﬁnally 3x 5 min with

500 µl anhydrous Acetone. All washing steps were done in the AFS2 at +2 °C. Washing steps were carried out by transferring the ﬂow-

through capsules with samples to 1.5 ml Eppendorf tubes containing 500 µl precooled washing solution. 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 (detailed protocol Appendix III).

Inﬁltration with Epon resin/Acetone mixtures was perfor¬med at 4 °C 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)

Inﬁltration schedule for 100% Epon was performed at room temperature:

pure Epon for 8 h (100% Epon)

pure Epon for 18 h (100% Epon)

pure Epon for 6 h (100% Epon)

Finally, ﬂow-through capsules containing carriers and Aclar discs with neurons were placed in 500 µl Eppendorf tubes containing 250

µl pure Epon, ﬁlled up with pure Epon and polymerized for 72 h at 65 °C. After polymerization ﬂow-through capsules were taken out of

the Eppendorf tubes and opened with a razorblade to take out the sample.The resin around the carrier was trimmed away after which

the carrier could be removed from the resin. Next the resin around the Aclar disc was trimmed away after which the Aclar disc could

be taken off with forceps leaving the neurons behind in the resin. Approximately 70 nm thick en face sections were cut on an ultrami-

crotome (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 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 0.375 nm).

Courtesy: Elly van Donselaar, Martin Harterink, Karin Vocking, Wally Müller and Casper Hoogenraad, Universiteit Utrecht, The Nether-

lands.

Reference:

Müller, M., Marti, T., and Kriz, S. 1980. Improved structural preservation by freeze- substitution.

Electron Microscopy,

Vol. 2, pp.

720–721, Brederoo, P., and De Priester W.(Eds.)

North Holland, Amsterdam, The Netherlands.

Appendix I

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.

Appendix II

Freeze-substitution medium:

1. Solution A: prepare 10 ml 0.6% (w/v) Uranyl Acetate in anhydrous Methanol (Merck, 1.06012) and next dissolve 200 mg OsO4.

Cool down this solution for 30 min at –90 °C in the AFS2.

2. Solution B: prepare 10 ml 6% (v/v) Glutaraldehyde in anhydrous Methanol (from 10% (v/v) GA stock in anhydrous Methanol (EMS,

16532)) and cool down this solution for 30 min at –90 °C in the AFS2.

3. Cool down 2.0 ml Sarstedt tubes for 30 min at –90 °C in the AFS2. Remove the rubber ring from the lids before cooling down

the tubes.

4. Cool down a 15 ml glass vial with snap or screw cap for 30 min at –90 °C in the AFS2.

5. Pipet equal volumes of precooled solution A and B in the precooled 15 ml glass vial, and mix well by pipetting up and down at

least three times.

6. Fill 2.0 ml Sarstedt tubes with 800 µl precooled FS medium

Appendix III

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 - CULTURED RAT HIPPOCAMPAL NEURONS

RESULTS

Fig. 2 Detail of Neurite. SV-Synaptic vesicles, Mt-Microtubules

Fig. 1 Detail of Soma. M-Mitochondria, ER-Endoplasmatic reticulum, G-Golgi

Leica Microsystems EM AFS2, EM ICE Application Note

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