+86(027) 5111 3188
This kit is a high-sensitivity two-step immunohistochemistry kit, suitable for subsequent color development of antibodies whose primary antibodies are derived from mice. The kit contains horseradish peroxidase HRP-conjugated goat anti-mouse IgG. This product is suitable for mouse-derived primary antibodies. In IHC and Western blot experiments, HRP-labeled goat anti-mouse secondary antibodies are combined with The mouse-derived primary antibody (reacts with the antigen first) is combined and then reacts with DAB. DAB is a substrate of horseradish peroxidase (HRP). Under the catalysis of HRP, DAB produces brown precipitates to achieve signal amplification and color development.
Storage and transportation
Transport in wet ice; store the secondary antibody at -20°C, and store the DAB dilution and 50×DAB stock solution at 4°C; the validity period is 12 months.
Ready to work
1. Bring your own PBS (recommended G4202, G0002), nuclear counterstaining reagents (recommended hematoxylin staining solution G1004, hematoxylin differentiation solution G1039, hematoxylin returning blue solution G1040), gradient alcohol, xylene, mounting tablets, etc.
2. Prepare the working solution of the secondary antibody: Dilute the HRP-labeled goat anti-mouse secondary antibody with PBST (pH 7.2-7.4) at a ratio of 1:200 to obtain the working solution of the secondary antibody. Prepared for immediate use, use within 48 hours.
3. Prepare DAB color developing working solution: add 20 μL of 50×DAB stock solution to every 1 mL of DAB diluent, mix well for later use. Prepared on demand, ready for use now.
1. According to the routine immunohistochemical procedures, the tissue sections are pre-deparaffinized, antigen retrieval, blocked, and incubated with the primary antibody, and then washed three times with PBS for 5 minutes each.
2. Secondary antibody incubation: Add 50-100 μL of the secondary antibody working solution to each slice to completely cover the tissue, and incubate at room temperature for 30-60 min. Wash three times with PBS for 5 minutes each time.
3. DAB color development: add 50-100 μL DAB working solution dropwise to each slice, incubate for several minutes at room temperature, observe under a microscope to control the color development to the expected effect, and then immediately wash with water to stop the color development.
4. (Optional) Restaining of cell nuclei: sections are stained with hematoxylin staining solution for 3-5 min, and then washed with tap water; after rapid differentiation with hematoxylin differentiation solution for 3-5 s, then washed with tap water; washed with tap water or returned to blue with hematoxylin solution 3-5 s.
5. Mounting: the slices are dehydrated by gradient alcohol according to the usual steps, xylene is transparent, and the slides are mounted with neutral gum.
1. If the background is too dark during immunohistochemical color development, consider extending the washing time, use an appropriate blocking solution for blocking, inactivate endogenous catalase, shorten the color development time, and reduce the concentration of the secondary antibody. If there is no color development or the color development is too light, you can appropriately increase the concentration of the primary antibody and the secondary antibody to extend the color development time, and then check whether the secondary antibody is normally developed.
2. DAB is harmful to the human body, please be careful when handling it and take care to avoid direct contact with the human body or inhalation of the body.