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Getting started with EnzMet™ is simple!
Just replace your diaminobenzidine (DAB) chromogenic color development reagent with the EnzMet™ HRP Detection Kit for IHC / ISH.
It really is that easy!. The same HRP conjugate will work just as well or better when you use EnzMet™ instead of DAB.
All the other steps and reagents stay the same, except perhaps for adjusting antibody dilutions.
EnzMet™ enzyme metallography: How it Works.
For best performance, here are a few pointers:
- EnzMet™ is frequently more sensitive than DAB. This means you can usually increase the dilution of the primary antibody for optimum results. In a study of a range of antigens, a 5-fold or 10-fold extra dilution was found to be about right for many markers; however, depending on your particular target and its distribution, you may find best results at the same dilution, or up to 50-fold extra. Start with a dilution 5-fold greater than that used for DAB, and adjust up or down as necessary.
- You can still use polymerized HRP probes with EnzMet™, or even other target amplification systems, for example tyramide signal amplification using biotin-tyramide/streptavidin detected with streptavidin-HRP or avidin-biotin-complex (ABC) methods with EnzMet™, although in most cases sensitivity will be just as high using simple, conventional monomeric immunoperoxidase probes.
- Nickel or other metal-based enhancement procedures should not be used with EnzMet™: these may affect the metallographic reaction or cause background.
- As with any immunostaining procedure, there are potential problems. We have covered some of these in our previous issue on EnzMet™, but here are a few more tips and tricks for immunohistochemical staining:
- Make sure that endogenous peroxidase activity is blocked with 3% hydrogen peroxide with a thorough rinsing before application of the immunoperoxidase reagent. Enhanced sensitivity applies to endogenous peroxidase as well.
- If background cannot be attributed to endogenous peroxidase, try reducing the concentration of the immunoperoxidase secondary reagent five or ten-fold and increasing the incubation time. If you still see background, the following treatments may be helpful:
- Washing with 0.02 to 0.1 M sodium citrate solution, pH 3.5.
- Include 0.1% Tween-20 in the wash buffer used immediately before rinsing and application of the EnzMet™ development reagent.
- Thorough rinsing with water before application of the EnzMet™ reagents: halide ions (such as the chlorides present in phosphate-buffered saline) can precipitate the metal ions in EnzMet™.
- If development continues after you wash off the EnzMet™ reagents, try some of the "stop" reactions used for silver enhancement. Washing 1-2 minutes with 1% freshly prepared sodium thiosulfate is frequently effective in these cases, or quench peroxidase activity with 3% hydrogen peroxide. For alternatives, check the advice on our technical support page for silver enhancement.
- If development has gone too far, you can reverse it using the same back-development procedure used for silver enhancement. Treat the over-developed specimen with Farmer's solution (0.3 ml 7.5% potassium ferricyanide, 1.2 ml of 20% sodium thiosulfate, 60 ml water) and monitor carefully until staining intensity is satisfactorily reduced.
- Tubbs R.; Pettay J.; Powell R.; Hicks D. G.; Roche P.; Powell W.; Grogan T., and Hainfeld, J. F.: High-resolution immunophenotyping of subcellular compartments in tissue microarrays by enzyme metallography. Appl. Immunohistochem. Mol. Morphol., 13, 371-375 (2005).
- Abstract from the Applied Immunohistochemistry and Molecular Morphology
- Powell, R. D.; Hainfeld, J. F.; Gutierrez, E.; Furuya, F. R.; Joshi, V. N.; Liu, W., and Takvorian, P. M.: Nanogold Labels, Covalent Gold, and Enzyme Metallography as Components of Nanodevices. AIP Conf. Proc. (Fritzsche, W., and Bier, F., Eds.), 1062, 91-99 (2008).
- Abstract from the AIP Conference Proceedings.
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