Electron microscope sample preparation technique_V2
Wai Kit Lam (Leo)
Abstract
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.
Attachments
Steps
Fixation
Aspirate cell culture media then fix cells with pre-warmed phosphate-buffered 4% Paraformaldehyde (PFA) at Room temperature for 1h 0m 0s.
Wash cells twice with 0.1Molarity (M) sodium-cacodylate (NaCac) 7.4 buffer.
Post-fix the sample with 2.5 glutaraldehyde in 0.1Molarity (M) NaCac buffer for 24h 0m 0s at 4°C.
Wash cells three times with 0.1Molarity (M) NaCac buffer.
Post-fix the sample with 1 osmium tetroxide (OsO4), 1.5 Potassium ferricyanide (K3Fe(III)(CN)6) in 0.1Molarity (M) NaCac buffer On ice for 1h 0m 0s.
Bring the sample to BioWave Pro microwave (Pelco), and expose it to three microwave dutycycle (0h 2m 0s on, 0h 2m 0s off) at 100W under vacuum.
Rinse cells three times with MilliQ water.
en bloc staining
Add 2 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.
Rinse cells three times with MilliQ water.
Agarose embedding
Scrape and pellet (10000x g) cells in MilliQ water.
Resuspend the cell pellet in 70% volume Ethanol.
Centrifuge the sample again at 10000x g.
Gently remove the supernatant (i.e. 70 % Ethanol).
Add one drop of low-melting point agarose to the cell pellet.
Vortex and centrifuge the sample immediately.
Cut the solidified agarose-cell pellet into 1 mm cubes using a razor blade.
Transfer the agarose-cell pellet cubes to a flat-bottom crew cap tube.
Dehydration
Perform a microwave assisted dehydration by graduated ethanol series (80 %, 90 %, 95 %, 100 %, 100 % (v/v); each at 150 W for 0h 0m 40s).
Perform a microwave assisted dehydration by propylene oxide (100%, 100% (v/v); each at 150 W for 0h 0m 40s).
Infiltration
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 0h 3m 0s under vacuum).
Resin embedding
Put the infiltrated agarose-cell pellet cubes and the appropriate paper label into the resin-block mould, then fill up the mould with resin.
Incubate the resin-embedded sample to a 60°C oven for 48h 0m 0s or until the resin is fully polymerised.
Resin-embedded sample trimming
Use a razor blade to carefully trim the resin block to expose the underlying agarose-cell pellet cubes.
Create a mirror surface on the agarose-cell pellet cubes using Ultra UCT ultramicrotome (Leica Biosystems) equipped with a glass knife.
Ultrathin sectioning
Cut 70-90 nm sections on an ultramicrotome equipped with a diamond knife (Ultra 45° Diatome).
Load the ultrathin sections onto the grid. (Copper side up)
The grid is ready for TEM imaging.
