Specificity: anti-Mouse IgG (H+L), anti-Rabbit IgG (H+L)
Enzyme: Alkaline Phosphatase
Chromogen/Substrate: Fast-Red/Naphthol Phosphate Buffer
TL-015-AF: 15mL Ultra V Block (TA-015-UB), 15mL Primary Antibody Enhancer (TL-015-PB), 15mL AP Polymer (TL-015-AP), 15mL Naphthol Phosphate Substrate (TA-015-AFS), three tablets 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 Primary Antibody Enhancer and incubate for 20 minutes at room temperature.
10. Wash 4 times in buffer.
11. Incubate with 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 an 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.