Specificity: anti-Mouse IgG (H+L), anti-Rabbit IgG (H+L)
Enzyme: Alkaline Phosphatase
Chromogen/Substrate: Fast Red/Naphthol Phosphate Buffer
TL-015-AFS: 15mL Ultra V Block (TA-015-UB), 15mL Value Primary Antibody Enhancer (TL-015-PBS), 15mL Value AP Polymer (TL-015-APS), 15mL Naphthol Phosphate Substrate (TA-015-AFS), 3 Fast Red Tablets (TA-003-AFC)
UltraVision detection system detects a specific mouse IgG or rabbit IgG antibody bound to an antigen in tissue sections. The specific antibody is located by a universal secondary antibody formulation conjugated to an enzyme-labeled polymer that recognizes mouse and rabbit immunoglobulins. The polymer complex is then visualized with an appropriate substrate/chromogen.
1. Deparaffinize and rehydrate tissue section.
2. Wash 2 times in buffer.
3. If required, incubate tissue in digestive enzyme (or appropriate pretreatment).
4. Wash 4 times in buffer.
5. 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. (May be omitted if primary antibodies are diluted in buffers containing 5-10% normal goat serum.)
6. Wash (Optional).
7. Apply primary antibody and incubate according to manufacturer's recommended protocol.
8. Wash 4 times in buffer.
9. Apply Value Primary Antibody Enhancer and incubate for 20 minutes at room temperature.
10. Wash 4 times in buffer.
11. Incubate with Value AP Polymer anti-Mouse/Rabbit IgG for 30 minutes at room temperature.
12. Wash 4 times in buffer.
13. Add 1 Fast Red tablet to 5mL of Naphthol Phosphate Substrate and shake until tablet is dissolved. Apply to tissue section and incubate for 10-20 minutes or until desired stain intensity is achieved.
14. Wash 4 times in DI water.
15. Counterstain and cover slip using aqueous mounting media.
The specificity and sensitivity of antigen detection is dependent on the specific primary antibody used. General References:
1. Shan-Rong Shi, James Guo, Richard J. Cote, Lillian Young, Debra Hawes, Yan Shi, Sandra Thu, and Clive R. Taylor, Applied Immunohistochemistry & Molecular Morphology, Vol 7, 201-208, 1999.
2. Karen Petrosyan, Rosalba Tamayo, and Daisy Joseph, “Sensitivity of a Novel Biotin-free Detection Reagent (PowerVision+) for Immunohistochemistry” J. Histotechnology, Vol 25, 247-250, 2002.