Specificity: anti-Mouse IgG (H+L), anti-Rabbit IgG (H+L)
Enzyme: Alkaline Phosphatase
Chromogen/Substrate: Fast-Red/Naphthol Phosphate Buffer
TL-015-AFJ: 15mL Ultra V Block (TA-015-UB), 15mL UltraVision ONE AP Polymer (TL-015-APJ), 15mL Liquid Fast-Red Chromogen (75X) and Naphthol Phosphate Substrate (TA-015-AL)
UltraVision One AP polymer detection system detects rabbit and mouse immunoglobulins bound to an antigen in tissue sections. The specific primary antibody is located by a universal secondary antibody polymer formulation. The amino acid polymer is conjugated to alkaline phosphatase and the Fab fragments of goat anti-rabbit and goat anti-mouse. The polymer complex is then visualized with an appropriate substrate/chromogen.
NOTE: The appropriate controls, especially negative controls, must be included with every manual or automated slide run. The inclusion of negative controls will aid in accurate interpretation of the staining results and help in determining false positives. Refer to the warnings and precaution section for details.
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. Optional: 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. Incubate with UltraVision One AP Polymer for 30 minutes at room temperature.
10. Wash 4 times in buffer.
11. Prepare the working solution liquid fast red immediately before use and use within 15 minutes of preparation or decreased sensitivity may result.
12. Add 1 drop (40μL) of Liquid Fast Red Chromogen to 3mL of Naphthol Phosphate Substrate and mix it well.
13. Apply solution to tissue section and ncubate tissue section for 10-25 minutes.
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.