Specificity: anti-Mouse IgG (H+L), anti-Rabbit IgG (H+L)
Chromogen/Substrate: 3-amino-9-ethylcarbazole (AEC)
TP-015-HA: 15mL Hydrogen Peroxide Block (TA-015-HP), 15mL Ultra V Block (TA-015-UB), 15mL Biotinylated Goat Anti-Polyvalent (TP-015-BN), 15mL Streptavidin Peroxidase (TS-015-HR), 15mL AEC Substrate (TA-015-HAS), 15mL AEC Chromogen (TA-002-HAC)
UltraVision detection system detects a specific antibody bound to an antigen in tissue sections. The specific antibody is located by a biotin-conjugated secondary antibody. This step is followed by the addition of a streptavidin-enzyme conjugate that binds to the biotin present on the secondary antibody. The specific antibody, secondary antibody, and streptavidin-enzyme complex is then visualized with an appropriate substrate/chromogen.
1. Deparaffinize and rehydrate tissue section.
2. To reduce nonspecific background staining due to endogenous peroxidase, incubate slide in Hydrogen Peroxide Block for 10-15 minutes.
3. Wash 2 times in buffer.
4. If required, incubate tissue in digestive enzyme (or appropriate pretreatment).
5. Wash 4 times in buffer.
6. Apply Ultra V Block and incubate for 5 minutes at room temperature to block nonspecific background staining. NOTE: Do not exceed 10 minutes or there may be a reduction in desired stain.
7. Rinse (Optional).
8. Apply primary antibody and incubate according to manufacturer's protocol.
9. Wash 4 times in buffer.
10. Apply Biotinylated Goat Anti-Polyvalent and incubate for 10 minutes at room temperature.
11. Wash 4 times in buffer.
12. Apply Streptavidin Peroxidase and incubate for 10 minutes at room temperature.
13. Rinse 4 times in buffer.
14. Add 20-50μL (1 drop) of AEC Chromogen to 1mL of AEC Substrate and mix well. Apply mixture to tissue section within 20 minutes of mixing. Incubate tissue section for 5-20 minutes, depending on the desired stain intensity.
WARNING: AEC is a suspected carcinogen. Handle with care and dispose of according to all regulations.
15. Counterstain and cover slip.
NOTE: AEC produces a red end product that is soluble in alcohol and must be used with an aqueous counterstain and mounting media.
The specificity and sensitivity of antigen detection is dependent on the specific primary antibody used.