Boster Immunoleader

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HRP Conjugated anti-Rabbit IgG SABC Kit

SA1028

1 kit

140 USD

HRP Conjugated anti-Rabbit IgG SABC Kit

SA1028

140 USD

1 kit

Ready to use (No dilution needed)

4˚C for one year. Avoid freezing.

IHC-F;ICC

SABC (StreptAvidin-Biotin Complex) is specially designed for displaying the distribution of antigens in tissues and cells in immunochemistry and other immunodetection analyses. Streptavidin is a 47,000 dalton protein purified from the bacterium Streptomyces avidinii. Streptavidin has extraordinarily strong affinity to biotin molecules. The dissociation constant (Kd) of the biotin-streptavidin complex is on the order of ~10-15 mol/L, a million times higher than the typical affinity between antigens and their antibodies. Streptavidin has very low non-specific binding to tissues and cells, due to its nearly neutral isoelectric point (IP=6.0~6.5). Therefore, immunohistochemical analyses based on streptavidin-biotin complex has extremely low background. Furthermore, this kit has high sensitivity because each complex it generates has a large number of peroxidase and streptavidin molecules. In brief, SABC offers high specificity, low background and ease-of-use.

1. 5% BSA Blocking Reagent: 12 ml, for blocking tissue sections. 2. Biotinylated Secondary Antibody (Mouse Anti-rabbit IgG): 12ml (10ug/ml). Affinity purified antibody, labeled with “long-arm” biotin (Biotinamidohexanoic acid N-hydroxysuccinimide ester, CAS# 72040-63-2). 3. SABC-POD (HRP conjugated streptavidin): 12ml (20ug/ml). Manufactured by Boster’s proprietary method, the complex is very stable and offers superior amplification of the antigen signals.

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).

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 rabbit.

Assay Procedure Blood smear, cultured cell and frozen section staining process 1. Cover the entire surface of a clean microslide with POLY-L-LYSINE (Catalog number: AR0003). Incubate for 1 minute then rinse the microslide with water.  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. 4. Add 5% BSA blocking reagent solution to the sample and incubate at room temperature for 20 minutes. Discard the blocking reagent solution, but do not wash the tissue section. 5. Add properly diluted primary antibody (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.) 6. Add biotinylated mouse anti-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. 7. 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. 8. 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. 9. 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. 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 sample. 2. If the direct staining result of frozen tissue sections is not satisfactory, the tissue sections may be repaired by the following procedure:  Before the 4th step above, heat the sample in 0.01M citrate buffer (pH6.0) to the boiling point with an electric heater or microwave oven, and then let the sample cool for 5~10 minutes. Repeat once. Wash the sample with 0.02 M PBS (pH 7.2~7.6) once or twice when it cools to room temperature.