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N A N O P R O B E S     N E W S
Vol. 11, No. 4          April, 2011


EnzMet™
Enzyme Metallography
Tips and Tricks

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Transitioning to EnzMet™ from DAB

Because staining with EnzMet™ is similar to using conventional enzyme chromogens such as DAB, moving from DAB to EnzMet™ is usually a very easy transition.

Transitioning from other enzymatic methods is easy.

With the exception of the replacement of the DAB with the EnzMet™ development reagents, a typical EnzMet™ protocol uses the same reagents and procedure as the DAB protocol.

However, that does not mean nothing can go wrong, so here are a few guidelines and modifications that will help if you have questions or encounter problems:

EnzMet™ is frequently more sensitive than the corresponding DAB detection reagent.

As a result you can increase the dilution of the primary antibody.

In a study of a range of antigens, we find that 5-fold or 10-fold extra dilution is about right for many markers; however, depending on the target and its distribution, additional dilutions may vary between nothing and 50-fold.

Start with a 5-fold additional dilution and adjust up or down as necessary.

 

EnzMet™ background reduction

Background is usually very low,
but if you do encounter any, the following may help:

  1. Washing with 0.02 to 0.1 M sodium citrate solution, pH 3.5.
  2. Addition of 0.1% Tween-20 to the wash buffer used immediately before rinsing and applying the EnzMet™ development reagent.
  3. Thorough rinsing with water before application of the EnzMet™ reagents: halide ions (such as the chlorides present in phosphate-buffered saline) can precipitate the metal ions in EnzMet™.

 

Check our technical support page for silver enhancement.

The chemistry of EnzMet™ is similar to our Silver Enhancer for Nanogold®; it responds to similar background reduction methods, “stop” reactions and back-development (to reverse development if it has gone too far) as silver enhancement, so the same methods are also appropriate for EnzMet™.

 

Osmication after EnzMet™

If you are considering an osmication step after EnzMet™ development, particularly if you are combining it with uranyl acetate, then etching of the EnzMet™ signal may be a risk: try reducing the osmium tetroxide concentration to 0.1% or gold toning if this is an issue.

 

 

More information:

 

 

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