May 24, 2023
Electron microscope sample preparation technique_V2
Forked from Electron microscope sample preparation technique
- Wai Kit Lam (Leo)1
- 1Laboratory of Michael Lazarou (Team Hurley) at Monash University
Protocol Citation: Wai Kit Lam (Leo) 2023. Electron microscope sample preparation technique_V2. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn75zmv5d/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 02, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 68051
Keywords: ASAPCRN, em sample preparation protocol, preparing em sample, sample preparation, processing cultured cell sample, cultured cell sample, em sample, preparation, sample trimming, embedded sample trimming, sample
Funders Acknowledgements:
Aligning Science Across Parkinson’s
Grant ID: ASAP-000350
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
Materials
List of reagents
- 0.2 M Phosphate buffer (pH 7.4)
- 0.2 M sodium-cacodylate (NaCac) (pH 7.4)
- 16 % Paraformaldehyde solution, EM grade
- 25 % Glutaraldehyde solution, EM grade
- 2 % osmium tetroxide (OsO4)
- Potassium ferricyanide (K3Fe(III)(CN)6)
- Uranyl acetate (UA)
- Lead citrate
- Agarose, low-gelling point
- 70 %, 80 %, 90 %, 95 %, 100 % Ethanol
- Propylene oxide
- Dodecenylsuccinic anhydride (DDSA)
- Araldite 502
- Procure 812
- Benzyldimethylamine (BDMA)
Troubleshooting
Safety warnings
For hazard information and safety warnings, please refer to the SDS (Safety Data Sheet).
Fixation
1h
Aspirate cell culture media then fix cells with pre-warmed phosphate-buffered 4% Paraformaldehyde (PFA) at Room temperature for 01:00:00 .
1h
Wash cells twice with 0.1 Molarity (M) sodium-cacodylate (NaCac) 7.4 buffer.
Post-fix the sample with 2.5 Mass Percent glutaraldehyde in 0.1 Molarity (M) NaCac buffer for 24:00:00 at 4 °C .
1d
Wash cells three times with 0.1 Molarity (M) NaCac buffer.
Post-fix the sample with 1 Mass Percent osmium tetroxide (OsO4), 1.5 Mass Percent Potassium ferricyanide (K3Fe(III)(CN)6) in 0.1 Molarity (M) NaCac buffer On ice for 01:00:00 .
1h
Bring the sample to BioWave Pro microwave (Pelco), and expose it to three microwave dutycycle (00:02:00 on, 00:02:00 off) at 100W under vacuum.
4m
Rinse cells three times with MilliQ water.
en bloc staining
Add 2 Mass Percent 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.
Note
It is best to filter the 2% (w/v) UA using 0.22 µm pore size filter.
Rinse cells three times with MilliQ water.
Agarose embedding
Scrape and pellet (10000 x g, 00:03:00 ) cells in MilliQ water.
Resuspend the cell pellet in 70 % volume Ethanol.
Centrifuge the sample again at 10000 x g, 00:03:00 .
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.
Note
The low-melting point agarose must be super-hot (close to 100°C) to achieve step 15.
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
40s
Perform a microwave assisted dehydration by graduated ethanol series (80 %, 90 %, 95 %, 100 %, 100 % (v/v); each at 150 W for 00:00:40 ).
40s
Perform a microwave assisted dehydration by propylene oxide (100%, 100% (v/v); each at 150 W for 00:00:40 ).
40s
Infiltration
3m
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 00:03:00 under vacuum).
Note
The composition of the Resin is 51.7 % (w/w) DDSA, 18.6 % (w/w) Araldite 502, 26.3 % (w/w) Procure 812 and 3.4 % (w/w) BDMA.
3m
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.
Note
It is important to remove all the bubbles. Usually, some bubbles are trapped under the paper label. It is best to use a pair of forceps to lift-up the paper label to release these bubbles, followed by using a 1 ml plastic transfer pipette to remove them.
Incubate the resin-embedded sample to a 60 °C oven for 48:00:00 or until the resin is fully polymerised.
2d
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.
Note
Before proceeding to the ultrathin sectioning, the block face must be cleaned, and the edges on the mirror surface must be parallel to each other.
Ultrathin sectioning
8m
Cut 70-90 nm sections on an ultramicrotome equipped with a diamond knife (Ultra 45° Diatome).
Note
Make sure you check the angle of the knife holder. For the Ultra 45° Diatome, the angle needs to be at 6°.
Load the ultrathin sections onto the grid. (Copper side up)
The grid is ready for TEM imaging.