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.
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 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 Reagent 3 (Normal goat serum) to the section. Incubate for 30 min at 37℃.
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℃ for 1~2h or at 4℃ overnight (then rewarm at 37℃ for 30 min). Wash with PBS or TBS, 2 min × 3.
7. Add Reagent 1, incubate at room temperature or 37℃ for 20 min. Wash with PBS or TBS, 2 min×3.
8. Add Reagent 2, incubate at room temperature or 37℃ for 20~30 min. Wash with PBS or TBS, 2 min×3.
9. Add 1 drop (approximately 50 μL) of Reagent 4 (DAB Concentrate) into each 1 mL of Reagent 5 (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.
Store at 2~8℃, shading light. Avoid of freezing. Valid for 12 months. The reagents are valid within 6 months after opening.
fSubmitted [ Dec 04 2018 ]
Asked: hi i want to ask if i want to use 2-step plus Poly-HRP Anti Rabbit/Mouse IgG Detection System(with DAB Solution) Catalog No: E-IR-R213 as secondary antibody and detection kit in small intestine tissue of rat ,i want to ask about size and quantity i require to prepare 100 slides using FFPE of IHC thank you
adminSubmitted [ Dec 04 2018 ]
Answered: If 50ul is added to completely cover tissue samples, 3ml E-IR-R213 can be used for 60 slices, the size of 6ml E-IR-R213 will be enough for your 100 slices. If 100ul is added to completely cover tissue samples, 10mL will be enough for your experiment, so our advice is that you can purchase two vials of 6mL E-IR-R213.
A*************iSubmitted [ Aug 06 2018 ]
Asked: Does this kit need any dilution since it certainly contains the secondary antibody? If yes what is the dikution ratio? And do you have a rough estimate of how many slides/slices this 3ml kit could serve? Thank you.
S**aSubmitted [ Aug 06 2018 ]
Answered: I have question about time for incubation DAB
D****************oSubmitted [ Mar 19 2018 ]
Asked: Can you please expatiate on Step 2: Retrieve antigen of the tissue section according to special requirements of applied primary antibody. I\'m working with Anti TNF-alpha for IHC on formalin fixed paraffin embedded tissues.
P*******nSubmitted [ Mar 19 2018 ]
Answered: Dear Dr. Margaret, Thanks for your question. As your samples are formalin fixed paraffin embedded tissues, it is suggested to repair paraffin embedded tissues(antigen) according to procedures provided in the manual.