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 goat. 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-R214A
|
5% BSA
|
3 mL
|
6 mL
|
18 mL
|
2~8°C
|
|
E-IR-R214B
|
Polyperoxidase-anti-Goat IgG (Ready-to-Use)
|
3 mL
|
6 mL
|
18 mL
|
2~8°C
|
|
E-IR-R214C
|
3% H2O2
|
3 mL
|
6 mL
|
18 mL
|
2~8°C
|
|
E-IR-R214D
|
DAB Concentrate (20×)
|
150 μL
|
300 μL
|
900 μL
|
2~8°C
|
|
E-IR-R214E
|
DAB Substrate
|
3 mL
|
6 mL
|
18 mL
|
2~8°C
|
|
Manual
|
|
One Copy
|
Sample dyeing
1. Dewax
and hydrate the paraffin section.
2. Make
thermal repair or digestion treatment to antigen of the tissue section if
necessary according to antigen/antibody situation.
3. Incubate with E-IR-R214C (3% H2O2)
for 10 min to eliminate endogenous peroxidase activity. Wash with PBS or TBS, 2
min× 3.
4. Add E-IR-R214A (5% BSA), incubate at room temperature for 30
min. Shake off any excess liquid.
5. Add primary antibody (Goat-IgG) with proper dilution ratio,
incubate at 20~37℃ for 1~2h or at 4℃ overnight (then rewarm at 37℃ for 30 min). Wash with
PBS or TBS, 2 min× 3. Dry the section with absorbent paper.
6. Add E-IR-R214B (Polyperoxidase-anti-Goat IgG), incubate at
room temperature or 37°C for 20 min. Wash with
PBS or TBS, 2 min×3.
7. Add 1 drop (approximately 50 μL) of E-IR-R214D (DAB Concentrate) into each 1 mL of E-IR-R214E (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.
8. Take control of the DAB coloration period, the
color of tan or brownish yellow is the positive signal. Avoid of excessive reaction.
9. 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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