TITLE: Electron microscope sample preparation technique_V2
AUTHORS: Wai   Kit Lam (Leo)

[DESCRIPTION]
There are eight main steps in preparing EM sample, including fixation, en bloc staining, agarose embedding, dehydration, infiltration, resin embedding, resin-embedded sample trimming and ultrathin sectioning.

This EM sample preparation protocol is established by Benjamin Padman and slightly modified by Wai Kit Lam and Runa Lindblom. Of note, this protocol is designed for processing cultured cell sample.

[STEPS]
SECTION: Fixation
1. Aspirate cell culture media then fix cells with pre-warmed phosphate-buffered 4% Paraformaldehyde (PFA) at Room temperature for 60 min.

SECTION: Fixation
2. Wash cells twice with 0.1 Molarity (M) sodium-cacodylate (NaCac) pH 7.4 buffer. 

SECTION: Fixation
3. Post-fix the sample with 2.5 1498 glutaraldehyde in 0.1 Molarity (M) NaCac buffer for 1440 min at 4 °C.

SECTION: Fixation
4. Wash cells three times with 0.1 Molarity (M) NaCac buffer.

SECTION: Fixation
5. Post-fix the sample with 1 1498 osmium tetroxide (OsO4), 1.5 1498 Potassium ferricyanide (K3Fe(III)(CN)6) in 0.1 Molarity (M) NaCac buffer on ice for 60 min.

SECTION: Fixation
6. Bring the sample to BioWave Pro microwave (Pelco), and expose it to three microwave dutycycle (2 min on, 2 min off) at 100W under vacuum.

SECTION: Fixation
7. Rinse cells three times with MilliQ water.
SECTION: en bloc staining
8. Add 2 1499 uranyl acetate (UA) in MilliQ water to sample, followed by exposing the sample to three microwave duty-cycle (120s on, 120 s off) at 100W under vacuum.
 


SECTION: en bloc staining
9. Rinse cells three times with MilliQ water.
SECTION: Agarose embedding
10. Scrape and pellet (10000 x g, 3 min) cells in MilliQ water.

SECTION: Agarose embedding
11. Resuspend the cell pellet in 70 % volume Ethanol.

SECTION: Agarose embedding
12. Centrifuge the sample again at 10000 x g, 3 min.

SECTION: Agarose embedding
13. Gently remove the supernatant (i.e. 70 % Ethanol).
SECTION: Agarose embedding
14. Add one drop of low-melting point agarose to the cell pellet.
SECTION: Agarose embedding
15. Vortex and centrifuge the sample immediately.
 

SECTION: Agarose embedding
16. Cut the solidified agarose-cell pellet into 1 mm cubes using a razor blade.
SECTION: Agarose embedding
17. Transfer the agarose-cell pellet cubes to a flat-bottom crew cap tube.
SECTION: Dehydration
18. Perform a microwave assisted dehydration by graduated ethanol series (80 %, 90 %, 95 %, 100 %, 100 % (v/v); each at 150 W for 40 s).

SECTION: Dehydration
19. Perform a microwave assisted dehydration by propylene oxide (100%, 100% (v/v); each at 150 W for 40 s).

SECTION: Infiltration
20. Infiltrate samples with Araldite 502/Procure 812 (Resin) by graduated concentration series in propylene oxide (25 %, 50 %, 75 %1 100 %, 100 % (v/v)); each at 250 W for 3 min under vacuum).
 


SECTION: Resin embedding
21. Put the infiltrated agarose-cell pellet cubes and the appropriate paper label into the resin-block mould, then fill up the mould with resin.
 

SECTION: Resin embedding
22. Incubate the resin-embedded sample to a 60 °C oven for 2880 min or until the resin is fully polymerised.

SECTION: Resin-embedded sample trimming
23. Use a razor blade to carefully trim the resin block to expose the underlying agarose-cell pellet cubes.
SECTION: Resin-embedded sample trimming
24. Create a mirror surface on the agarose-cell pellet cubes using Ultra UCT ultramicrotome (Leica Biosystems) equipped with a glass knife.
 

SECTION: Ultrathin sectioning
25. Cut 70-90 nm sections on an ultramicrotome equipped with a diamond knife (Ultra 45° Diatome).
 

SECTION: Ultrathin sectioning
26. Load the ultrathin sections onto the grid. (Copper side up)
SECTION: Ultrathin sectioning
27. The grid is ready for TEM imaging.