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.
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-R215A
|
Normal Goat Blocking
Buffer (Ready-to-Use)
|
3 mL
|
6 mL
|
18 mL
|
2~8°C
|
|
E-IR-R215B
|
Polyperoxidase-anti-Rabbit IgG (Ready-to-Use)
|
3 mL
|
6 mL
|
18 mL
|
2~8°C
|
|
E-IR-R215C
|
3% H2O2
|
3 mL
|
6 mL
|
18 mL
|
2~8°C
|
|
E-IR-R215D
|
DAB Concentrate (20×)
|
150 μL
|
300 μL
|
900 μL
|
2~8°C
|
|
E-IR-R215E
|
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-R215C (3% H2O2)
for 10 min to eliminate endogenous peroxidase activity. Wash with PBS or TBS, 2
min× 3.
4.Add E-IR-R215A (Normal Goat Blocking
Buffer (Ready-to-Use)), incubate at 37°C for 30 min. Shake off any
excess liquid.
5.Add
primary antibody (Rabbit-IgG) with proper dilution ratio, incubate at 20~37°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. Dry the section with absorbent paper.
6.Add
E-IR-R215B (Polyperoxidase-anti-Rabbit 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-R215D (DAB Concentrate) into each 1 mL of E-IR-R215E
(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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