Thermo Scientific™

Lab Vision™ MultiVision Polymer Detection System: anti-Mouse-HRP and anti-Rabbit-AP

 Related applications:
Anatomical Pathology
Employ a simple and robust one-step polymer detection system for visualization of two antigens simultaneously in blue and red with the Thermo Scientific™ Lab Vision™ MultiVision™ Polymer Detection System.

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Description

MultiVision AP and HRP polymers are innovative, patented technologies consisting of smaller amino acid polymer subunits that minimize conflicts in binding the target protein. The smaller polymer allows for decreased binding conflicts, resulting in more consistent staining and better signal amplification.
  • A primary antibody cocktail of choice may be used in conjunction with ready-to-use MultiVision polymer cocktail, composed of anti-mouse/ horseradish peroxidase (HRP) plus anti-rabbit/alkaline phosphatase (AP)
  • Cocktail is specially formulated from MultiVision polymers that provide increased sensitivity, time-savings, and detection simplicity
  • Yields higher sensitivity and antibody efficiency as well as better signal-to-noise ratios
  • Biotin-free, eliminating background staining
  • Antigen Specificity: anti-Mouse IgG (H+L) and anti-Rabbit IgG (H+L)

  • Enzyme: Peroxidase and Phosphatase

  • Chromogen/Substrate: LVBlue/ LVRed

Includes:

TL-012-MHRA: 1 MultiVision anti-rabbit/AP + anti-mouse/HRP polymer (cocktail) (TL-012-RAM), 1 Ultra V block (TA-012-UB), 1 Hydrogen Peroxidase Block (TA-012-HP), 1 LVBlue AP Buffer (TA-075-TMM), 1 LVBlue Chromogen Vial 1 (TA-001-AB1), 1 LVBlue Chromogen Vial 2 (TA-001-AB2), 1 LVBlue Chromogen Vial 3 (TA-001-AB3), 1 LVRed Chromogen Vial 1 (TA-004-HR1), 1 LVRed Chromogen Vial 2 (TA-002-HR2), 1 LVRed Chromogen Vial 3 (TA-002-HR3), 1 LVRed Chromogen Vial 4 (TA-002-HR4), 1 Mounting Media (TA-005-UA)

Note:

MultiVision Polymer Detection System may be employed with any primary antibody cocktail to visualize the presence of two proteins simultaneously. After the specific antibodies are bound to their respective antigens in tissue sections, the rabbit and mouse primary antibodies are located by a specially formulated cocktail of secondary antibody polymers: anti-mouse/ horseradish peroxidase (HRP) + anti-rabbit/alkaline phosphatase (AP). In general, the amino acid polymer is conjugated to AP or HRP enzymes and the Fab fragments of goat anti-rabbit or goat anti-mouse immunoglobulins. The polymer complex is then visualized with first the LVBlue chromogen for AP activity and after washing with the LVRed chromogen for HRP activity. A weak hematoxilin nuclear counterstain is not recommended.

WARNING: Reproductive Harm - www.P65Warnings.ca.gov
Procedure (MANUAL USE):

STAINING PROTOCOL (kit components in bold)

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. Paraffin tissue sections: deparaffinze in xylene substitute and rehydrate in graded alcohols.

2. Wash 2 times in TBST buffer: (TBS + 0.1% Tween™ 20).

3. Perform tissue pretreatment: heat-induced epitope retrieval for 20 min at 98°C followed by cooling at room temperature for 20 minutes.

4. Wash 4 times with DI water.

5. Apply Hydrogen Peroxidase Block solution and incubate 10 minutes at room temperature.

6. Wash 4 times in TBST.

7. Apply Ultra V Block and incubate for 10 minutes at room temperature to block nonspecific background staining. NoteDo not exceed 10 minutes or there may be a reduction of staining intensity.

8. Do not wash after Ultra V Block, but blot off the blocking solution and continue with next step.

9. Apply primary antibody cocktail and incubate for 30 minutes at room temperature.

10. Wash 4 times in TBST.

11. Incubate with MultiVision Polymer Cocktail: anti-mouse/ horseradish peroxidase (HRP) + anti-rabbit/ alkaline phosphatase (AP) for 30 minutes at room temperature.

12. Wash 4 times in TBST.

13. Prepare working solution of LVBlue immediately before use and use within 15 minutes of preparation or decreased sensitivity may result.

14. Add 1 drop of LVBlue vial 1 to 2.5mL of AP-buffer and mix well.

15. Add 1 drop of LVBlue vial 2 to AP-buffer and mix well.

16. Add 1 drop of LVBlue vial 3 to AP-buffer and mix well.

17. Apply solution to tissue section and incubate for 10 minutes.

18. Wash 4 times in TBST.

19. Prepare working solution of LVRed immediately before use and use within 15 minutes of preparation or decreased sensitivity may result.

20. Add 3 drops of LVRed vial 1 to 5mL of DI water and mix well.

21. Add 2 drops of LVRed vial 2 to mixture and mix well.

22. Add 2 drops of LVRed vial 3 to mixture and mix well.

23. Add 2 drops of LVRed vial 4 to mixture and mix well.

24. Apply solution to tissue section and incubate for 10 minutes.

25. Wash 4 times in DI water.

26. Dry tissue section completely, preferably on a 60°C hotplate for one hour.

27. Mount with xylene-substitute containing medium provided with the kit. NOTE 1: Use of aqueous mounting media will make the LVRed reaction product disappear in a few days. NOTE 2: Use of xylene-containing mounting medium may partly dissolve the LVBlue reaction product. Procedure (FOR AUTOSTAINER):

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. Paraffin tissue sections: deparaffinze in xylene-substitute and rehydrate in graded alcohols.

2. Wash 2 times in TBST buffer.

3. Perform tissue pretreatment: heat-induced epitope retrieval for 20 minutes at 98°C followed by cooling at room temperature for 20 minutes.

4. Wash 4 times with DI water.

5. Apply Hydrogen Peroxidase Block solution and incubate 10 minutes at room temperature.

6. Rinse with TBST.

7. Apply Ultra V Block and incubate for 10 minutes at room temperature to block nonspecific background staining. Note: do not exceed 10 minutes or there may be a reduction of staining intensity.

8. Apply primary antibody cocktail and incubate for 30 minutes at room temperature.

9. Rinse with TBST.

10. Incubate with MultiVision Polymer Cocktail: anti-mouse/ horseradish peroxidase (HRP) + antirabbit/alkaline phosphatase (AP) for 30 minutes at room temperature.

11. Rinse with TBST.

12. Prepare fresh working solution of LVBlue immediately before use (follow steps 14 – 16, above). Apply and incubate for 10 minutes using the Substrate-Batch feature.

13. Rinse 3 times; once with TBST, followed by DI water and TBST, respectively.

14. Prepare fresh working solution of LVRed immediately before use (follow steps 20 – 23 above). Apply and incubate for 10 minutes using the Substrate-Batch feature.

15. Rinse 4 times with DI water.

16. Dry tissue section completely, preferably on a 60°C hotplate for one hour.

17. Mount with Xylene-substitute mounting medium provided in the kit. NOTE 1: Use of aqueous mounting media will make the LVRed reaction product disappear in a few days. NOTE 2: Use of xylene-containing mounting medium may partly dissolve the LVBlue reaction product. General References:

1. Immunoenzyme Multiple Staining Methods. CM van der Loos, Handbook no. 45, BIOS Scientific Publishers, Oxford, UK, 1999. ISBN: 1-85996-187-8 or Springer-Verlag, New York, ISBN: 0-387-91594-x.

2. Multiple staining in molecular morphology, CM van der Loos. In: Molecular morphology in human tissues (Eds. Hacker GW and Tubbs RR), CRC Press, Boca Raton, FL, 2005, pp. 27-63.



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