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Product Introduction:
This kit is suitable for immunohistochemical test of rabbit IgG with DAB color development.
SABC is designed for immunohistochemical and other immune assays to show the distribution of antigens in tissues and cells. StreptAvidin is a protein derived from Streptomyces and has a high affinity for biotin like avidin, 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 is Streptavidin-biotin Complex. SABC can form a complex composed of about 100 peroxidase and about 50 StreptAvidin, and a large number of enzymes will ensure high sensitivity of SABC.
Operation steps: (Take paraffin section as an example)
1. Routine dewaxing of slices to water (three times xylene, three times ethanol).
2, 3% H2O2 Room temperature for 5-10 minutes to inactivate endogenous peroxidase, washed in distilled water for 3 times.
3. Optional steps:
A. Heat repair antigen, dip the slice into 0.01M sodium citrate buffer (pH 6.0), 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 (pH 7.2-7.6).
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, drop 5% BSA sealing liquid, room temperature 20 minutes, discard excess liquid, free wash.
5. Dilute the primary antibody with diluent in a certain proportion (the diluted primary antibody can be stored at 4℃ for a week), or you can purchase the antibody diluent separately. Drop and dilute the primary antibody incubated at 37℃ for about 1 hour or 20℃ for about 2 hours, or 4℃ overnight. 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).
6. According to the dosage, dilute Bio-Sheep anti-Rabbit IgG concentrate with diluent at 1:50-100 into working liquid (1mL diluent is mixed with 10-20μL Bio-sheep anti-mouse IgG concentrate, and 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, 2 minutes each time.
7. According to the amount of use, the SBC-POD concentrate is diluted by 1:100 into the working liquid (1mL dilution is mixed with 10μL SBC-POD concentrate, and the working liquid can be stored at 4℃ for one week). Add the SBC-POD working solution and incubate at 20-37℃ for 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 50μL 20×DAB color development solution A and 50μL 20×DAB color development solution B according to 1mL of PBS. After fully mixed, add drops to the slice, color development at room temperature, and control the reaction time under the mirror, 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, before DAB color development, wash the slices 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.
Note：Product information may be optimized and upgraded. Please refer to the actual label information for accuracy.
