Benchtop Preparation for TEM

Transmission electron microscopy (TEM) can be a fantastic way to obtain a wealth of information about biological structures and processes. Unfortunately the conditions inside a TEM are not particularly hospitable for biological tissues. To successfully pass electrons through TEM specimens, they must be thinner than a wavelength of visible light, resistant to hard vacuum and completely dehydrated. (In other words: dead!)

Preparing biological specimens for TEM therefore requires preservation, dehydration and embedding of the sample for sectioning on an ultramicrotome. Specimen preparation is arguably the most important factor in acquiring high quality images, so here is a generic (“benchtop”) protocol that we use as a guide when preparing samples for ultra-thin sectioning. See below the protocol for important notes... but first...

DISCLAIMER: This protocol is meant strictly as a guide for persons with a high level of technical competency. I do not provide a warranty of any kind for the quality of specimens prepared using it, and I cannot be held responsible in any way. Do not use this guide if you are unqualified or unsure. Read the MSDS for all chemicals and reagents before use, and use the correct personal protective equipment. Fear osmium, because it can kill you.

Generic Benchtop Specimen Preparation:

Step	Reagent	Time
Primary Fixation	4% PFA in Buffer	1 hour
Secondary Fixation	2.5% GA in Buffer	1 hour
3-5x Buffer Wash	0.1M Buffer	10 min
Tertiary Fixation	1% OsO4 (aq) in Buffer	1 hour
1-2x Buffer Wash	0.1M Buffer	10 min
3x Water Wash	MilliQ Water	10 min
En Bloc Staining	2% Uranyl Acetate	1 hour
3x Water Wash	MilliQ Water	10 min
Dehydration	50% Ethanol	10 min
Dehydration	70% Ethanol	10 min
Dehydration	90% Ethanol	10 min
Dehydration	100% Ethanol	10 min
Dehydration	100% Ethanol	10 min
Dehydration	100% Transition Solvent	15 min
Dehydration	100% Transition Solvent	15 min
Infiltration	Solvent:Resin (3:1) [25%]	1 hour
Infiltration	Solvent:Resin (1:1) [50%]	1 hour
Infiltration	Solvent:Resin (1:3) [75%]	1 hour
Infiltration	Resin 100%	1.5 hour
Embedding	Resin 100% @ 60-70°C	24 hour

Common acronyms:

PFA = Paraformaldehyde

GA = Glutaraldehyde

OsO₄ = Osmium Tetroxide

Reagent Note:

The buffers, transition solvents and resins are often tailored to specific projects. A good starting point would be 0.1M Phosphate Buffer, Acetone and Epon-Araldite (aka. Procure-Araldite).