rev: May 14, 2008
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Immunoasaay Enhancers
for Researchers and Manufacturers
READY TO USE TMB FOR HISTOCHEMISTRY & HYBRIDIZATION APPLICATIONS
-see also TMB-PPT for use with aqueous based reagents and for blotting use
catalog#RDI-TMBSH-1L, new cat#: 42R-TB103 $484.00/liter
cat# RDI-TMBSH, new cat#:
42R-TB103x $275.00/500ml
USE: For the Immunohistochemical Localization
of Peroxidase Labeled Probes May also be Employed for
in situ Hybridization and Blotting Procedures Stable up to 18 months at
room temp or 24 months at 4 DEG C.
Background: Tetramethylbenzidine (TMB) {3,3',5,5'Tetramethylbenzidine} is an excellent substrate for
detecting horseradish peroxidase (HRP) labeled
probes. TMB reagents for ELISA and blotting techniques have been available for
several years. Histochemically, TMB has been utilized
primarily for the study of retrograde axonal transport. To prevent needle
formation during color development, sodium nitroprusside
in combination with a low pH was used. Recent publications have described
stabilization of the TMB precipitate with ammonium hexamolybdate
or sodium tungstate. Methyl salicylate
has also been employed to render the TMB reaction product insoluble in
alcohols.
This new RDI product is a NEW, stable-single component TMB for histochemical use containing none of these stabilizers and
enhancers. The final reaction product is not affected by ethanol, xylene or xylene substitutes.
Thus dehydration through alcohols to xylene and
mounting the sections in a xylene type medium is
possible. The product may also be used for blotting procedures.
Method: After reacting with a HRP labeled probe, tissue sections or
blots are incubated in the TMB solution. A one electron oxidation occurs
resulting in the formation of a stable blue precipitate at the sites of HRP
activity.
REAGENTS PROVIDED
Tetramethylbenzidine Solution: Contains TMB, 1.25 mMol L(-1) in an acetate buffer, pH 4.9. Also contains proprietary stabilizers and precipitating agents.
Store at room temperature or store at 4-8 DEG C, bring to room temp before use.
Discard if solution is turbid or blue
Avoid exposure to direct sunlight, iron and copper salts and bleach
Neutral Red Solution:(not included, available spearately) Contains Neutral Red, 0.025% buffered at pH 5.1 Store at room temperature Discard if a precipitate forms
SAMPLE PROCEDURE: (must be optimized for each application):
1. Add primary antibody and incubate sections (blots for an ap propriate interval.
2. Wash sections in a buffer such as PBS containing 0.05% Tween
20.
3. Block endogenous peroxidase, if necessary, by
incubating sections in absolute methanol at room temperature.
4. Wash briefly in PBS.
5. Incubate sections for 30 minutes at room temperature in PBS containing 0.03%
hydrogen peroxide.
6. Wash sections in PBS-Tween-20.
7. Add peroxidase labeled probe and incubate for an
appropriate interval
NOTE: The HRP probe must be titered. For example, a 1:50 dilution of an HRP conjugate that performs well for DAB must be diluted 1:800 for use with TMB.
8. Wash sections in PBS-Tween-20.
9. Cover sections with TMB solution and incubate 5-15 minutes.
10. After TMB incubation is complete, stop reaction by incubating 10 minutes in
deionized water.
11. Counterstain 30-60 seconds in Neutral Red
Solution.
12. Rinse in deionized water the
dehydrate rapidly through alcohols to xylene.
13. Mount sections using a xylene based medium.
RESULTS
Site of HRP activity will be brilliant blue.
For In Vitro Research or Further Manufacturing Use Only
RDI Division of Fitzgerald Industries Intl
phone (978) 371-6446 or (800) 370-2222
fax (978) 371-2266
Email:antibodies@fitzgerald-fii.com
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