Boster Immunoleader

other

DyLight®488 Conjugated Human IgG SABC Kit

SA1098

1 kit

350 USD

DyLight®488 Conjugated Human IgG SABC Kit

SA1098

350 USD

1 kit

Concentrated (Need dilution)

4˚C for one year. Avoid freezing.

IHC-P;IHC-F;ICC

IHC(P): 1:200-800 IHC(F): 1:200-800 ICC: 1:200-800 Optimal dilutions should be determined by end users.

DyLight is a new type of fluorescent dye and has been widely used in recent years. The antibodies conjugated by it is brighter than that conjugated by Cy2 and FITC because of its better spectral width, higher tolerance of optics, stronger specificity, less sensitive to pH and better permeability. The DyLight®488-SABC system brings the streptavidin - biotin into fluorescent system and can further enhance the sensitivity and suppress the background. The emission and filtration wavelength of DyLight®488 are 493 nm and 518 nm respectively.

1. Normal goat serum blocking reagent: 10x, 10 ml, for the block of tissue sections. 2. Biotinylated Secondary Antibody (Rabbit Anti-human IgG): 100x, 1 ml (2mg/ml). Affinity purified antibody, labeled with “long-arm” biotin (Biotinamidohexanoic acid N-hydroxysuccinimide ester, CAS# 72040-63-2). 3. SABC-DyLight®488 (DyLight®488 conjugated streptavidin): 200~800x, 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. 0.01M 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. 0.1% trypsinase or the compound digest solution (Catalog number: AR0022).

Human 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 human.

Assay Procedure A. Paraffin section 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. To heat repair the antigen, soak the tissue section in 0.01M citrate buffer (pH 6.0), and heat to the boiling point with an electric heater or a microwave oven, then stop heating. Repeat this heating process 2~3 times with a 5~10-minute interval. Wash the tissue section with distilled water 3 times for 2 minutes each. 4. Dilute normal rabbit serum blocking reagent at 1:10 with 0.01M PBS (pH7.2-7.6). Add the diluted 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. 5. Add properly diluted primary antibody (human 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 0.01M PBS (pH 7.2~7.6) 3 times for 2 minutes each. 6. Add biotinylated rabbit anti-human IgG (diluted at 1:100 with 0.01M PBS (pH 7.2~7.6)) to the tissue section and incubate at 20~37˚C for 30 minutes. Wash the section with 0.01 M PBS 3 times for 2 minutes each. 7. Dilute SABC-DyLight®488(Streptavidin-DyLight®488) at 1:200-800 with 0.01M PBS (pH 7.2~7.6). Add the diluted SABC-DyLight®488 solution to the tissue section and incubate at 20~37˚C for 30 minutes. Wash the tissue section 4 times with 0.01M PBS (pH 7.2~7.6) for 5 minutes each. 8. Put several drops of the water soluble sealing reagent onto the tissue section and seal with a cover slide. The tissue section is ready for observation under a fluorescence microscope. B. Blood smear, cultured cells and frozen section 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 3 times for 2 minutes each. 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 4-8 in the heat repair antigen process.