UA-Zero® EM Stain is a patented solution developed as a direct replacement for Uranyl Acetate. UA-Zero does not contain any radioactive material and is non-toxic. UA-Zero can be used as a direct substitute for Uranyl Acetate with no changes to standard user protocols.

UA-Zero is supplied in an opaque dropper bottle, which should be stored long-term in the lab fridge at around 4°C. For daily use, the sealed bottle can be stored in normal laboratory conditions away from direct sunlight.

It has been shown that UA-Zero EM Stain works very well for various protocols that specify Uranyl Acetate as a staining solution for TEM sample preparation in life science. This application note describes one example of a protocol for preparing samples and subsequent imaging using TEM. Uranyl Acetate and UA-Zero EM stain were directly compared and the images generated are shown in the Results section.

Staining the sample on a TEM grid, with a support film, requires just a few simple steps before drying.

Protocol used for TEM sample preparation

All steps performed at room temperature unless otherwise stated. Parts of the same sample have been stained using UA-Zero EM Stain and Uranyl Acetate, for direct comparison.

Fixation
1.     2.5% glutaraldehyde (AGR1012) in 0.1M cacodylate bufferDays**
2.     0.1M cacodylate (AGR1105) washes3 x 10 mins
3.     1% OsO(AGR1021) in 0.1M cacodylate buffer1 hour
4.     0.1M cacodylate washes 3 x 10 mins
5.     Deionised water2 x 5 mins
Staining
6a.   UA-Zero EM Stain in 20% ethanol1 hour
6b.   3% Uranyl Acetate in water1 hour
7.     Deionised water2 x 10 mins
Dehydration
8.     70% ethanol10 mins
9.     80% ethanol10 mins
10.   90% ethanol10 mins
11.   100% ethanol3 x 20 mins
12.   Propylene oxide2 x 5 mins
Infiltration
13.   Propylene oxide: 'Epon' Resin mix*, 1:13.5 hours
14.   'Epon' Resin MixOvernight
15.   'Epon' Resin Mix~ 6 hours
Embedding
13.   In 'Epon' Resin cure at 60°C2 days

Sections cut and picked up on pioloform copper slot grids. No further staining of sections.

*‘Epon’ type resin mix:

  • 24g AGR1045 Agar 100 Resin
  • 16g AGR1053 DDSA (Dodecenyl Succinic Anhydride) EM grade redistilled
  • 10g AGR1083 MNA (Methyl Nadic Anhydride) redistilled
  • 1.5g AGR1062 BDMA (Benzyl Dimethylamine)

** Length of fixation time not known precisely as the sample was prepared from a control tissue used for another study (can be days to weeks, tissue stored in the fixation solution at 4°C).

Results

TEM images of sample prepared using UA-Zero EM Stain in 20% ethanol as staining solution:

TEM images of sample prepared using 3% Uranyl Acetate in water as staining solution:

Samples prepared and imaged courtesy of Prof. Dr. Paul Verkade and Dr. Chris Neal, Wolfson Bioimaging Facility, School of Medical Sciences, University of Bristol.