Introduction
2-step plus is a two-step immunohistochemical broad spectrum detection reagent. It polymerizes monovalent Fab fragments of secondary antibody and enzyme, which replaces the secondary antibody and tertiary antibody in traditional method, can directly amplify the binding signal of antibody-antigen. This method not only retains the specific binding ability of antibody with antigen, but also can effectively avoid space steric hindrance caused by excessive polymer molecules. Compared with the traditional SP three-step method, this kit has the characteristics of simple, rapid, high-sensitivity. This system abandons the using of biotin, so it can avoid background staining by endogenous biotin. It can be used in IHC, in which the primary antibody is monoclonal/polyclonal antibody derived from rabbit/mouse. The unique polymer auxiliary agent for macromolecular detection system can detect the binding primary antibody better.
Diluent for DAB concentrated solution has been provided in this kit to avoid the influence of the different water
acidity and alkalinity on the DAB Chromogenic Agent.
Components
|
Cat
|
product
|
3 mL
|
6 mL
|
18 mL
|
Storage
|
|
E-IR-R213A
|
3% H2O2
|
3 mL
|
6 mL
|
18 mL
|
2~8°C
|
|
E-IR-R213B
|
Polymer Helper (Ready-to-Use)
|
3 mL
|
6 mL
|
18 mL
|
2~8°C
|
|
E-IR-R213C
|
Polyperoxidase-anti-Mouse/Rabbit IgG (Ready-to-Use)
|
3 mL
|
6 mL
|
18 mL
|
2~8°C
|
|
E-IR-R213D
|
Normal Goat Blocking
Buffer (Ready-to-Use)
|
3 mL
|
6 mL
|
18 mL
|
2~8°C
|
|
E-IR-R213E
|
DAB Concentrate (20×)
|
150 μL
|
300 μL
|
900 μL
|
2~8°C
|
|
E-IR-R213F
|
DAB Substrate
|
3 mL
|
6 mL
|
18 mL
|
2~8°C
|
|
Manual
|
|
One Copy
|
Sample dyeing
1. Dewax and hydrate the paraffin section.
2. Repair antigen of the tissue section according to special requirements
of applied primary antibody.
3. Incubate with E-IR-R213A (3% H2O2) for 10 min to eliminate endogenous peroxidase
activity.
4. Wash with PBS or TBS, 2 min× 3.
5. Drain the PBS with
absorbent paper, then add E-IR-R213D (Normal Goat Blocking
Buffer) to the section.
Incubate for 30 min at 37°C.
6. Dry the liquid around the
section with absorbent paper, and draw a circle around the tissue with an oily
pen.Add primary antibody with proper dilution ratio, incubate at room
temperature or 37°C for 1~2h or at 4°C overnight (then rewarm at 37°C for 30
min). Wash with PBS or TBS, 2 min × 3.
7. Add E-IR-R213B (Helper), incubate at room temperature or 37°C for 20 min. Wash with PBS or TBS, 2 min×3.
8. Add E-IR-R213C(Polyperoxidase-anti-Mouse/Rabbit IgG), incubate at room temperature or 37°C for 20~30 min. Wash with PBS or TBS, 2 min×3.
9.Add 1 drop (approximately 50 μL) of E-IR-R213E (DAB Concentrate) into each 1 mL of E-IR-R213F(DAB Substrate), mix fully and the mixed reagent is the DAB Working
Solution.Prepare fresh solution before use and the prepared
solution should be stored in the dark.Fresh prepared DAB Working Solution is
valid within 4 hours and the unused solution must be abandoned.
10. Take control of the DAB coloration period, the color of
tan or brownish yellow is the positive signal. Avoid of excessive reaction.
11. Wash
the section with deionized water terminate the chromogenic reaction,
then operate the procedures of counterstaining, dehydrating, transparentizing and sealing.
Storage
Store at 2~8°C,
shading light. Avoid of freezing. Valid for 12 months. The reagents are valid
within 6 months after opening.
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