Product Introduction:

This kit is suitable for immunohistochemical test of rabbit IgG source. FITC fluorescence detection. SABC is designed for immunohistochemical and other immune assays to show the distribution of antigens in tissues and cells. StreptAvidin (StreptAvidin) is a protein extracted from Streptomyces, like avidin, has a high affinity for biotin, avidin is an alkaline protein (IP=10), and can be modified to become a neutral protein. The isoelectric point of streptavidin is close to neutral, and the non-specific adsorption to tissues and cells is very low. The background of streptavidin based immunohistochemical method is very low. SABC, or StreptAvidin - Biotin Complex, can form a complex of about 100 FITCs and about 50 streptavidins. A large number of FITCs will guarantee high sensitivity of SABC.

Operation steps: (trace reagents please centrifuge after use)

Take hot repair of paraffin section as an example

1. Routine dewaxing of slices to water.

2. Optional steps: A. Hot repair antigen, dip the slice into 0.01M sodium citrate buffer (PH6.0), heat in electric oven or microwave oven to boil, power off, and repeat 1-2 times after an interval of 5-10 minutes. After cooling, wash 1-2 times with PBS (pH7.2-7.4). b, enzyme digestion, drop digestive solution, 37℃10min, PBS (pH7.2-7.4) washing 2-3 times. c. Skip this step and go to the next step.

3. Add 5% BSA sealer and leave at room temperature for 20 minutes. Discard excess liquid and leave to wash.

4. Dilute the primary antibody with diluent in a certain proportion (the diluted primary antibody can be stored at 4℃ for one week), or you can purchase the antibody diluent separately. Diluted primary antibody was added and incubated at 37℃ for about 1h or 20℃ for about 2h, or overnight at 4℃. Wash with PBS (pH7.2-7.4) for 3 times, 2min each time (the dilution of primary antibody, incubation time and temperature are directly related to the staining intensity and background. Generally speaking, when the positive staining intensity is not enough, the concentration of primary antibody can be increased and the incubation time can be prolonged. When the background is too high, it can reduce the concentration of primary antibody and shorten the incubation time).

5. According to the amount of use, dilute the Bio-Sheep anti-Rabbit IgG concentrate by 1:100 into the working liquid (1ml dilute with 10ul Bio-sheep anti-rabbit IgG concentrate, mixed well to form the working liquid, the working liquid can be stored at 4℃ for one week). Bio-sheep Anti-Rabbit IgG working liquid was added and incubated at 20-37℃ for 30 minutes. Wash with PBS (pH7.2-7.4) for 3 times, 2min each time.

6. According to the amount of use, the SBC-FITC concentrate is diluted with diluent at 1:100 to the working liquid (1ml diluent is added to 10ul SBC-FITC concentrate and mixed to form the SBC-FITC working liquid, which can be stored at 4℃ for one week). Add SBC-FITC working solution and incubate at 20-37℃ for 30min(away from light). Wash with PBS (pH7.2-7.4) for 4 times, 5min each time.

7. Add anti-fluorescence attenuation sealant to seal the film. Fluorescence microscope observation. For cell crawling tablets, after fixation, rinsed twice with PBS, incubated with 0.5% Trxiton X-100 at room temperature for 20min, rinsed twice with PBS, followed by step 3 above. For frozen slices, after fixation, rinse twice with PBS, then follow step 2 above.

Note:

If the background was too high, after the SABC reaction, the slices were washed 4 times with PBST (pH7.2-7.4) with 0.01%-0.02% TWEEN-20, washed 2 times with PBS, and then sealed for observation.

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