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The following paper appeared in Microsc. Microanal., 10, (Suppl. 2: Proceedings) (Proceedings of Microscopy and Microanalysis 2004); Anderson, I. M.; Price, R.; Hall, E.; Clark, E., and McKernan, S., Eds.; Cambridge University Press, New York, NY, 2004, p. 1210CD.

Enzymatic Metallography as a Correlative Light and Electron Microscopy Method

F. R. Furuya,* V. N. Joshi.,* J. F. Hainfeld,* R. D. Powell,* and P. M. Takvorian**

* Nanoprobes, Incorporated, 95 Horseblock Rd., Yaphank NY 11980
** Biological Sciences Department, Rutgers University, Newark, NJ 07102

Chromogenic enzymatic staining is a well-established method for the imunohistochemical and cytochemical demonstration of antigens at the light microscope level [1]. Although enzymatic staining has been used for immunoelectron microscopy, the contrast between the deposited reaction product and the surrounding specimen is often less than desirable; this may be improved by nickel enhancement, but diffusion of the products may limit resolution, and the continuous nature of the staining may obscure the underlying cellular structure [2]. We have previously demonstrated that horseradish peroxidase-conjugated probes, treated with a mixture of an appropriate activating agent and a metal source, can selectively deposit metal at the target site [3]. In contrast to chromogenic substrates, metallographic staining gave a punctate pattern with very low cellular background.

Using control slides, washing with Lugols iodine, sodium periodate, or 1 % sodium thiosulfate were found to be most helpful in reducing background. In the staining of Her-2 protein in sections cut from a paraffin-embedded multiblock with positive and negative Her-2 control tissues, detection using the EnVision enzyme-polymer system (Dako) was found to give increased selectivity compared with direct staining using a HRP-conjugated secondary antibody (Figure 1). The higher atomic number of the metal, and the absence of diffusion implied by the staining pattern, suggested that the new reagents may also be useful for electron microscopy. This was tested in post-embedding labeling experiments with the microsporida Brachiola algerae; sporoblasts were labeled using a primary anti-polar tube (PT) protein primary antibody, followed by a secondary HRP conjugate developed for one minute with a tenfold dilution of the metallographic substrate. Staining was observed in locations consistent with the production of PT protein in the sporoblast [4]; in a control in which primary was omitted, staining was largely absent, although some background deposition was evident. (Figure 2). The results show that the new enzyme metallography procedure is effective for both light and electron microscopy, thus providing a simple correlative microscopy method.

In immunoblot tests, 1 ng of a biotinylated IgG target immobilized on a nitrocellulose membrane was detected using streptavidin-HRP with enzyme metallography; identical sensitivities were found using Nanogold-labeled streptavidin with silver enhancement (Figure 3), but background was lower with the enzyme metallography system. Preliminary tests were also made using the new enzyme metallography reagent to detect capture lines of an anti-steptococcus antibody printed into nitrocellulose strips; the results are shown in Figure 3(c) in comparison with the current latex-labeled antibody and with Nanogold with silver enhancement. The enhancement in sensitivity using enzyme metallography is dramatic, with relatively little background signal.


  1. A. G. Farr and P. K. Nakane: J. Immunol. Methods., 47 (1981) 129.

  2. E.-L. Punnonen et al: J. Histochem. Cytochem., 47 (1999) 99.

  3. J. F. Hainfeld et al: Microsc. Microanal., 8, (Suppl. 2: Proceedings); Lyman, C. al (Eds.); Cambridge University Press, New York, NY (2002) 916CD.

  4. A. Cali and P. M. Takvorian, J. Euk. Microbiol., 48 (2001) 82S.

  5. The authors thank Richard Eisen and Edison Narvaez, HT for helpful discussions and for providing specimens. This work was supported by SBIR grant 1R43 GM64257-01 from NIGMS, and STTR grant 2R42 CA83618-02 from NCI (NIH).
[Figures 1-3] (136k)
Figure 1: Staining of Her-2 protein in positive control tissue using polyclonal rabbit anti-human CerbB2 primary with (a) HRP-labeled secondary and DAB; (b) Envision enzyme-polymer conjugate with enzyme metallography (400 X magnification; bar = 25 micrometers).

Figure 2: (a) Brachiola algerae (microsporida associated with opportunistic infection in humans) reacted to an antibody to polar tube followed by HRP-labeled secondary and metallographic substrate, diluted tenfold. The sporoblasts show staining with similar distribution to known PT distribution; (b) control with pimary antibody omitted.

Figure 3: (a), (b) Immunoblot detection of biotinylated IgG, serial tenfold dilutions from 100 1 ng (upper row, left to right) and 1,000 1 pg (lower row, left to right), using (a) Streptavidin-HRP and enzyme metallography and (b) Nanogold-streptavidin with silver enhancement; (c) Antistreptococcal monoclonal capture antibody printed onto test strips and detected using Top: Protein A 300 nm latex conjugate; Middle: Nanogold anti-Mouse with silver enhancement; and Bottom: Polymerized anti-mouse-HRP dextran (Envison) with enzyme metallography.

Thanks to Cambridge University Press for allowing us to reproduce this online.
© 2004 Cambridge University Press. Used with permission.

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