Boster Immunoleader

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HRP Conjugated anti-Rabbit IgG Super Vision Assay Kit

SV0002-2

100 ml/kit

1000 USD

HRP Conjugated anti-Rabbit IgG Super Vision Assay Kit

SV0002-2

1000 USD

100 ml/kit

Ready to use (No dilution needed)

4˚C for one year. Avoid freezing

IHC-P;IHC-F;ICC

SV (Super Vision) Two-step Immunohistochemistry Assay Kit is researched independently by Boster Biological Technology, Ltd. Using polymerization marking method, HRP are conjugated with secondary antibody to form huge octopus-like molecule antibody-enzyme polymer. Thus, secondary antibody and third antibody in traditional method can be replaced by the enzyme polymer. And the enzyme polymer is specially fit for immunohistochemistry analyses for its powerful nature of amplifying signal and permeating tissues and cells. Comparing with troditional SABC kits (StreptAvidin-Biotin Complex), SV Kits possess superiorities of high speed, high sensitivity, low background and ease-of-use.

1. 5% BSA Blocking Reagent: 100ml, for blocking tissue sections. 2. Secondary Antibody (polymerization HRP conjugated anti-rabbit IgG): 100ml. 3. 3%H2O2: 100ml.

Rabbit IgG refers to the animal origin of the primary antibody, not the origin of the specimen.

Assay Procedure A. Immunohistochemistry paraffin tissue sections staining 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 or conduct digestive treatment to the antigen according to the state, 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 to room temperature. 5. Add 5% BSA blocking reagent solution to the tissue section and incubate at room temperature for 10 minutes. Discard the extra blocking reagent solution, but do not wash the tissue section. 6. Add properly diluted primary antibody (mouse 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 polymerization HRP marking anti-mouse IgG to the tissue section and incubate at37˚C for 30 minutes. Wash the tissue section with PBS or TBS 3 times for 2 minutes each. 8. DAB color development: Add 25-50mg DAB, 0.03% H2O2 to 100ml 0.02M PBS or TBS; Or 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. B. Blood smear, cultured cells or frozen sections staining process 1. Treat a microslide with POLY-L-LYSINE as described in Process A. ⑴ Blood samples. Add anticoagulant to the samples and smear the blood samples onto the treated microslide. ⑵ Cultured cells. Cultured cells can be smeared onto or directed cultivated on the treated microslide. ⑶ 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 acetone for 10~20 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 steps 4-9 in the immunohistochemistry paraffin tissue sections staining process. 4. Follow steps 5-9 in the immunohistochemistry paraffin tissue sections staining process.