MASTER DUAL STAINING KIT

Available Size : 100 test

Intended Use : Diagnostic in vitro in humans

Description: Master Dual Staining Kit contains reagents to perform manual or automated immunohistoquemical dual
staining on human tissue sections, fixed in buffered formalin and embedded in paraffin.
This kit contains a visualization system based on micropolymers and is sufficient to perform 100 determinations
following the recommended protocol.

Limitations Of The Reactives
The use on frozen tissue has not been evaluated.
In order to use the Master Dual Staining kit it should be noted that the two antibodies must come from different
animals (mouse / rabbit).
Due to the presence of a soluble chromogen in alcoholic solutions, the slides must be air-dried and for final mounting
an aqueous mounting media should be used.

Category:

Description & Usage

Principle Of The Analytical Method
The aim of the immunohistochemical staining is to convert into a visible stain the resulting antigen-antibody unit to be
studied in cells or tissues.
Thus, the Master Dual Staining Kit provides a set of highly specific and sensitive reagents which allow dual staining of
two different antigen-antibody units.
The kit is based on the use of a mix of two micropolymers, each labeled with different enzymes (Peroxidase and
Alkaline Phosphatase) each recognizing a specific immunoglobulin (antibody) developed in rabbit and mouse
respectively.
In case the reaction has occurred between the primary antibodies and antigens, the micropolymers specifically bind to
the resulted antigen-antibody unit. Due to enzyme label of the polymers, adding chromogen and its corresponding
substrates, precipitates of different colors are obtained which allows to microscopically detect the presence of a
specific antigens.
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Rev.: 2017-09-12
In the case of Master Dual Staining Kit, brown precipitates will be obtained for the antibodies developed in mouse
and red / purple color for rabbit antibodies.

Components and Reagents Included In The Kit:
• Peroxidase Blocking Reagent         MAD-021540Q-10        10mL
• POLYMER MIX                             MAD-001882QK-C        10mL

DAB:
DAB Substrate Buffer                     MAD-001812QK-A        15mL
DAB Chromogen Concentrate          MAD-001812QK-B        0,5mL

RED:
AP Substrate Buffer                       MAD-001818QK-A       2x15mL
AP Chromogen Concentrate            MAD-001818QK-B       0,5mL

1. Dewaxing and heat induced antigen retrieval2
a. Incubate slides with paraffin tissue sections in the oven at 60 ° C overnight.
b. Place the slides in the PT module using the flowing conditions:
– Pre-heat at 65ºC
– Dewax than followed by antigen retrieval 20 minutes a 95ºC

2. Detection and visualization (manual or automatic)
a. Wash in TBS – Tween 20 at RT
b. Apply 100 μl of Peroxidase Blocking Reagent on the tissue and incubate 10 min at RT
c. Wash 3 times in TBS.
d. Apply 100 μl of 1º Antibody* and incubate for 10 minutes** at RT
e. Wash 3 times in TBS.
f. Apply 100 μl of 2º Antibody* and incubate for 10 minutes** at RT
g. Wash 3 times in TBS.
h. Apply 100 μl of POLYMER MIX and incubate for 30 minutes at RT.
i. Wash 3 times in TBS.                                                                                                                                                               j. Mix a drop of DAB Chromogen Concentrate with 1 ml of DAB Substrate Buffer and applying the resulting
solution onto the tissue; incubate for 5 min at RT
k. Wash 3 times in distilled water
l. Mix a drop of AP Chromogen Concentrate with 2,5 ml of AP Substrate Buffer and applying the resulting
solution onto the tissue; incubate for 10 min at RT
m. Wash 3 times in distilled water

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