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Product Introduction:
This kit is suitable for DAB color development in immunohistochemical experiment of mouse IgG with primary antibody. 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, so the background of streptavidin based immunohistochemical method is very low. SABC, or StreptAvidin - Biotin Complex, can form a complex of about 100 peroxidase and about 50 streptavidin. A large number of enzymes will ensure that SABC is highly sensitive.
Operation steps: (Take paraffin section as an example)
1. Routine dewaxing of slices to water (three times xylene, three times ethanol).
2. 3% H2O2 at room temperature for 5-10 minutes to inactivate endogenous enzymes. Wash with distilled water 3 times.
3. Optional steps:
A. Heat repair antigen, dip the slice into 0.01M sodium citrate buffer (PH6.0), heat in electric oven or microwave oven to boil, then power off, interval 5-10 minutes, repeat 1-2 times. After cooling, wash 1-2 times with PBS (pH7.2-7.6).
b, enzyme digestion, drop digestive solution, 37℃ for 10 minutes, PBS (pH7.2-7.4) washing 2-3 times.
c. Skip this step and go to the next step.
4. Add 5% BSA sealer and leave at room temperature for 20 minutes. Discard excess liquid and leave to wash.
5. 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. Add diluted primary antibody and incubate at 37℃ for about 1 hour or 20℃ for 2 hours, or 4℃ overnight. Wash with PBS (pH7.2-7.4) for 3 times, 2 minutes each time. The dilution, incubation time and temperature of the primary antibody 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, the primary antibody concentration can be reduced and the incubation time shortened.)
6. According to the dosage, the Bio-Sheep anti-mouse IgG concentrate was diluted by 1:100 into the working liquid (1ml dilution was mixed with 10ul Bio-sheep anti-mouse IgG concentrate, and the working liquid could be stored at 4℃ for one week). Bio-sheep Anti-mouse IgG working liquid was added and incubated at 20-37℃ for 30 minutes. Wash with PBS (pH7.2-7.4) for 3 times, 2 minutes each time.
Seven. According to the amount of use, the SBC-POD concentrate was diluted by 1:100 into the working liquid (1ml dilution was mixed with 10ul SBC-POD concentrate, and the working liquid could be stored at 4℃ for one week). Add SBC-POD working solution,20-37℃, 30 minutes. Wash with PBS (pH7.2-7.4) 4 times for 5 minutes each time.
8. DAB color development: Prepared with PBS (pH7.2-7.4) according to the 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 slices. Room temperature color development, under the mirror control of the reaction time, generally between 5-30 minutes. Distilled water washing terminates the reaction.
9. Mild hematoxylin reinfection. Dehydrated, transparent, sealed. Microscope observation. For cell crawling tablets, after fixation, rinsed twice with PBS, incubated with 0.5% Triton X-100 at room temperature for 20 minutes, rinsed twice with PBS, and treated with 3% H2O2 for 15 minutes; Rinse twice with PBS and follow Step 4 above. For frozen slices, after fixation, rinse twice with PBS, then follow the second step above.
Note:
If the dyeing background is too high, after the 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.
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