rev:June 6, 2000
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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 $250.00/500ml $438.00/1 Liter $375.00/Liter 5-9
BULK ON REQUEST
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 Divison of researchd Industries Intl
San Jose, 95123 CA Snell ave 658
USA
or 408-780-0908
Email:margaret@cellular-research.com
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