Specificity: anti-Mouse IgG (H+L), anti-Rabbit IgG (H+L)
Chromogen/Substrate: Diaminobenzidine (DAB)
TL-015-HDJ: 15mL Ultra V Block (TA-015-UB), 15mL Hydrogen Peroxide Block (TA-015-HP), 15mL UltraVision ONE HRP Polymer (TL-015-PHJ), 15mL DAB Plus Substrate (TA-015-HSX), 1mL DAB Plus Chromogen (TA-001-HCX)
UltraVision ONE HRP 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 horseradish peroxidase 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. To reduce nonspecific background staining due to endogenous peroxidase, incubate slide in Hydrogen Peroxide Block for 10-15 minutes.
6. Wash 4 times in buffer.
7. 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.)
8. Wash (Optional).
9. Apply primary antibody and incubate according to manufacturer's recommended protocol.
10. Wash 4 times in buffer.
11. Apply UltraVision ONE HRP Polymer and incubate for 30 minutes at room temperature. (NOTE: HRP Polymer is light sensitive. Please avoid unnecessary light exposure and store in opaque vial.)
12. Wash 4 times in buffer.
13. Add 1 drop (40μL) DAB Plus Chromogen to 2mL of DAB Plus Substrate, mix by swirling and apply to tissue. Incubate for 5-15 minutes, depending on the desired stain intensity.
14. Wash 4 times in DI water.
15. Counterstain and cover slip using a permanent 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.