Cy3 Conjugated anti-Rat IgG SABC Kit

SABC (StreptAvidin-Biotin Complex) is specially designed for displaying the distribution of antigens in tissues and cells in immunochemistry and other immunodetection analyses. This kit has high sensitivity because each complex it generates has a large number of Cy3 and streptavidin molecules. Compared to the traditional immunodetection using free Cy3 dyes, the SABC-Cy3 system greatly enhances the sensitivity and suppresses the background. Cy3 is activated at 554nm, fluorescing at 568ca.574nm with bright red.

1. APES or POLY-L-LYSINE.
2. 0.02M PBS (pH 7.2ca.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).

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. Paraffin section staining process
Applies to immunohistochemical analysis of paraffin-embedded sections.
B. 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. 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 (ca.5um thick) with the treated microslide and bake in an oven at 58-60C 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 if there is a need, 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 2ca.3 times with a 5ca.10-minute interval. Wash the tissue section with distilled water 3 times for 2 minutes each.
4. Dilute the normal rabbit serum blocking reagent at 1:10 with 0.02M 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 (rat IgG) to the tissue section and incubate at 37C for about 1 hour or 20C for about 2 hours or at 4C overnight. Wash the tissue section with 0.02M PBS (pH 7.2ca.7.6) 3 times for 2 minutes each.
6. Add biotinylated rabbit anti-rat IgG (diluted at 1:100 with 0.02M PBS (pH 7.2ca.7.6)) to the tissue section and incubate at 20ca.37C for 30 minutes. Wash the section with 0.02 M PBS 3 times for 2 minutes each.
7. Dilute SABC-CY3 (Streptavidin-CY3) at 1:100 with 0.02M PBS (pH 7.2ca.7.6). Add the diluted SABC-CY3 solution to the tissue section and incubate at 20ca.37C for 30 minutes. Wash the tissue section 4 times with 0.02M PBS (pH 7.2ca.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 60ca.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.

4C for one year. Avoid freezing