Tetramethylbenzidine (TMB) staining

Henk Veldman H.Veldman at lab.azu.nl
Tue Sep 22 09:15:36 EST 1998

Louis Hom wrote:
> We're using TMB to stain cells labeled with antibody-HRP.  The
> signal/noise ratio is great, but we're having a problem mounting our
> specimens.  They all seem to cause rapid fading:  glycerol, nail polish,
> DPX (Aldrich), TBS's Tissue Freezing Medium, Permount, TissueTek's OCT.
> Any ideas? Most of them seem to be poly-ols, maybe that's a problem.
> Maybe a silicon-based mountant?  TIA.
> --
> ______________________________________________________________________________
> Lou Hom >K'93
> lhom at ocf.berkeley.edu
> http://www.ocf.berkeley.edu/~lhom/

We mount TMB-stained sections in DPX and never encounter any problems
with fading.
The dehydration prior to mounting might be critical: passing through
graded ethanols should be done quickly (70%, 95%, 100%, 100%, 10 sec
each on to xylene twice 2-5 min), but dehydration still should be
If complete dehydration cannot be acheived quickly this way, try
COMPLETE drying in air, xylene dip and mounting in DPX. Replacing
ethanol 100% with acetone might also do the trick.
BTW most of the *mountants* you name are not really mountants for
microscopy. In many cases were most mountants cause fading of a stain,
immersion-oil can be tried, this also works on most plastic substrates;
of course cover glasses should then be immobilized at the edges with
nail varnish or DPX or another suitable *glue*.

Hope this will be of help. Success .


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