Boster Immunoleader

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HRP Conjugated anti-Mouse/Rabbit IgG SABC Kit

SA2010

1 kit

300 USD

HRP Conjugated anti-Mouse/Rabbit IgG SABC Kit

SA2010

300 USD

1 kit

Concentrated (Need dilution)

4˚C for one year. Avoid freezing.

IHC-P;IHC-F;ICC

IHC(P): 1:50-200 IHC(F): 1:50-200 ICC: 1:50-200 Optimal dilutions should be determined by end users.

1. Normal goat serum blocking reagent: 10x, 10ml, for the block of tissue sections. 2. Biotinylated Secondary Antibody (Goat Anti-mouse IgG and Goat Anti-rabbit IgG): 100~200x, 1ml (2mg/ml) each. Affinity purified antibody, labeled with “long-arm” biotin (Biotinamidohexanoic acid N-hydroxysuccinimide ester, CAS# 72040-63-2). 3. SABC-POD (HRP conjugated streptavidin): 100~200x, 1ml (2mg/ml). Manufactured by Boster’s proprietary method, the complex is very stable and offers superior amplification of the antigen signals. 4. Three drop bottles (For dilution use).

1. APES or POLY-L-LYSINE. 2. 0.02M PBS (pH 7.2~7.6): 8.5g sodium chloride, 2.8g anhydrous Na2HPO4 and 0.4g anhydrous NaH2PO4 in 1000ml of distilled water. (The weight should be adjusted accordingly if hydrous phosphates are used.) 3. 0.01 M Citrate Buffer: 3g sodium citrate dihydrate (C6H5Na3O7•2H2O) and 0.4g citric acid monohydrate (C6H8O7•H2O) in 1000ml of distilled water. 4. DAB Chromogenic Kit (Catalog number: AR1022 or AR1025). 5. 0.1% trypsinase or the compound digest solution (Catalog number: AR0022).

Mouse/rabbit IgG refers to the animal origin of the primary antibody, not the origin of the specimen. This kit must be used on primary antibodies from mouse/rabbit.

Options of immunohistochemistry staining process The best process among the following may have to be identified by trial and error. The characteristics of the antigen/antibody used may be used as a guideline. A. Heat repair antigen process Applies to immunohistochemical analysis of paraffin-embedded sections, to expose the antibody binding site on the antigens. B. Enzyme digestion process Applies to immunohistochemical analysis of paraffin-embedded sections, to expose the antibody binding site on the antigens. C. Non-digestion/non-repair process Applies to stable antigens using immunohistochemical analysis of paraffin-embedded sections. D. Blood smear, cultured cells and frozen section staining process Applies to immunocytochemistry of blood smear and cultured cells, and immunohistochemical analysis of frozen-embedded sections. Assay Procedure A. Heat repair antigen process 1. Cover the entire surface of a clean microslide with APES (Catalog number: AR0001) or POLY-L-LYSINE (Catalog number: AR0003). Incubate for 1 minute then rinse the microslide with water. Mount a tissue section (~5um thick) with the treated microslide and bake in an oven at 58-60 ˚C for 30-60 minutes to ensure strong adhesion of the tissue section. 2. Dewax the tissue section in dimethylbenzene for 10 minutes and rinse with water. 3. Incubate the tissue section for 5~10 minutes in the 3% H2O2 solution to quench the endogenous peroxidase activity. Wash the tissue section with distilled water 3 times for 2 minutes each. 4. To heat repair the antigen, soak the tissue section in 0.01M citrate buffer (pH6.0), and heat to the boiling point with an electric heater or a microwave oven, then stop heating. Repeat this heating process 1~2 times with a 5~10-minute interval. Wash the tissue section with 0.02 M PBS (pH 7.2~7.6) once or twice when it cools down. 5. Add normal goat serum blocking reagent to the tissue section and incubate at room temperature for 20 minutes. Discard the blocking reagent solution, but do not wash the tissue section. 6. Add properly diluted primary antibody (mouse/rabbit IgG) to the tissue section and incubate at 37 ˚C for about 1 hour or 20 ˚C for about 2 hours or at 4 ˚C overnight. Wash the tissue section with 0.02M PBS (pH 7.2~7.6) 3 times for 2 minutes each. (The primary antibody concentration, incubation time and temperature directly affect the staining efficiency and background intensity. If the positive staining is too weak, the concentration of the primary antibody and the incubation time can be increased; if the background is too high, the primary antibody concentration and the incubation time can be decreased. ) 7. Add biotinylated goat anti-mouse/rabbit IgG to the tissue section and incubate at 20~37˚C for 20 minutes. Wash the tissue section with 0.02M PBS (pH 7.2~7.6) 3 times for 2 minutes each. 8. Add SABC-Peroxidase (Streptavidin-Peroxidase) to the tissue section and incubate at 20~37˚C for 20 minutes. Wash the tissue section 4 times with 0.02M PBS (pH 7.2~7.6) for 5 minutes each. 9. Use a DAB chromogenic kit (Catalog number: AR1022 or AR1025) to stain the tissue section. Add Reagent A, B and C, one drop each, into 1 ml of distilled water and mix thoroughly. Add this solution to the tissue section and incubate at room temperature. Control the time of incubation under a microscope. Usually 5~30 minutes is sufficient. Wash the tissue section with distilled water. 10. Slightly counterstain the tissue section with haematoxylin(if use AR1022) or nuclear fast red(if use AR1025), and wash with distilled water to clean the haematoxylin. Then dry the tissue section by baking, and put on a drop of resin seal the tissue section with a cover slide. The tissue section is ready for observation under a microscope. B. Enzyme digestion process The enzyme digestion process is similar to the heat repair antigen process. Simply replace the 4th step in the heat repair antigen process with the following.  Incubate the tissue section in 0.1% trypsinase or compound digestive solution (Catalog number: AR0022) for 5~10 minutes. Wash with distilled water 3 times. C. Non-digestion/non-repair process The process is for antigens which do not need heat repair or digestion. Simply omit the 4th step in the heat repair antigen process. D. Blood smear, cultured cells or frozen sections staining process 1. Treat a microslide with POLY-L-LYSINE as described in Process A.  Blood samples. Add anticoagulant to the samples and smear the blood samples onto the treated microslide.  Cultured cells. Cultured cells can be smeared onto or directed cultivated on the treated microslide.  Sections of frozen tissue. Sections of frozen tissue may be placed onto the treated microslide and air-dry at room temperature for 30 minutes until no liquid water is visible. 2. Fix the sample with 4% paraformaldehyde or 10% formalin for 60~90 minutes. 3. Dilute 30% H2O2 at 1:50 with pure methanol. Incubate the fixed sample for 30 minutes in the diluted H2O2 to quench the endogenous peroxidase activity. Wash the sample with distilled water once or twice. If the direct staining result of frozen tissue sections is not satisfactory, the tissue sections may be repaired by following the 4th step in the heat repair antigen process. 4. Follow steps 5-10 in the heat repair antigen process. Note 1. If the staining background is too high, wash the section with 0.01-0.02% TWEEN 20 PBS (pH7.2-7.6) 4 times and with pure PBS twice after SABC reaction and before DAB staining, then use DAB chromogenic kit to stain the section. 2. 0.01M citrate buffer (pH 6.0), PBS, or TBS buffer may be used to repair the section.