Product Introduction:

This kit is suitable for immunohistochemical experiments in which the primary antibody is mouse IgG. DAB and FITC color development. 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 very high affinity for biotin, and avidin is an alkaline protein (PI=10) that 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 peroxidase or FITC and about 50 streptavidin. A large number of enzymes and FITC will ensure that SABC is highly sensitive.

Operation steps: (Take hot repair of paraffin section as an example)

1. Routine dewaxing of slices to water. 3%H₂O₂ Room temperature for 5-10 minutes to inactivate endogenous enzymes (skip this step if FITC). Wash with distilled water 3 times.

2. Heat repair antigen: Dip the slices into 0.01M citrate buffer (PH6.0), heat in electric oven or microwave oven to boil, then power off, and repeat 1-2 times after 5-10 minutes. After cooling, wash 1-2 times with PBS (pH7.2-7.6).

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. Drop diluted primary antibody, incubate at 37 ℃ for 1 hour or 20℃ for 2 hours, or overnight at 4℃. PBS (pH7.2-7.4) was washed 3 times for 2 minutes each time (the dilution, incubation time and temperature of the primary antibody were 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-mouse IgG concentrate at 1:100 into the working liquid (1ml dilute with 10ul Bio-sheep anti-mouse IgG concentrate, mixed well to form the working liquid, the working liquid can be stored at 4℃ for one week). Biotinized sheep anti-mouse IgG working liquid was added and treated at 20-37℃ for 30 minutes. Wash with PBS (pH7.2-7.4) for 3 times, 2 minutes each time. If viewing with fluorescence, please follow steps 6 and 7, if using DAB for color development, please go directly to step 8.

6. According to the amount of use, dilute the SBC-FITC concentrate by 1:100 into the working liquid (1ml dilute and 10ul SBC-FITC concentrate are added, and then mix well to form the SBC-FITC working liquid. This working liquid can be stored at 4℃ for one week). Add the SBC-FITC working liquid at 20-37℃ for 30 minutes (avoid light). Wash with PBS (pH7.2-7.4) 4 times for 5 minutes each time.

7. Add anti-fluorescence attenuation sealant to seal the film. Fluorescence microscope observation.

8. According to the amount of use, dilute the SBC-POD concentrate by 1:100 into the working liquid (add 10ul SBC-POD concentrate to 1ml diluent and mix well to form the SBC-POD working liquid. This working liquid can be stored at 4℃ for one week). Drop the SBC-POD working liquid at 20-37℃ for 30 minutes. Wash with PBS (pH7.2-7.4) 4 times for 5 minutes each time.

9. DAB color development: Prepared with PBS (pH7.2-7.4) according to dosage, add 50ul 20×DAB color development solution A and 50ul 20×DAB color development solution B according to 1ml of PBS, mix well and add to slice. Room temperature color development, under the mirror control of the reaction time, generally between 5-30 minutes. Distilled water washing terminates the reaction.

10. Mild hematoxylin reinfection. Dehydrated, transparent, sealed. Microscope observation.

For cell crawling tablets:

After routine fixation, rinse twice with PBS, then use 0.5%Txiton X-100 at room temperature for 20 minutes, rinse twice with PBS, 3%H₂O₂ (if you use FITC, just skip this step) for 15 minutes; Rinse twice with PBS, then follow step 4 above. For frozen sections: After fixation, rinse twice with PBS, and then follow the second step above.

Note:

If DAB staining background is too high, after SABC reaction and before DAB color development, the slices are washed with PBST (pH7.2-7.4) added with 0.01-0.02% TWEEN-20 for 4 times, PBS for 2 times, and then DAB color development. If the background observed with FITC is too high, after the SABC reaction, the slices are washed with PBST (pH7.2-7.4) added with 0.01-0.02% TWEEN-20 for 4 times, and PBS for 2 times, and then the slices are sealed for observation. Because there are many immunohistochemical indicators and different specimens, this step is for reference only.

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