The Graham and Karnovsky Horseradish Peroxidase Ultrastructural Method: A Premier JHC Citation Classic

Abstract

This commentary briefly reviews the background for the development of the horseradish peroxidase–diaminobenzidine tetrahydrochloride histochemical method originally described by Graham and Karnovsky in their citation classic, reprinted in full in this issue of Journal of Histochemistry & Cytochemistry. Some of the method’s subsequent applications, including its use as a macromolecular tracer for kidney glomerular permeability and use in immunoelectron microscopy and other immunoassays, are also discussed.

The landmark paper by Richard Graham and Morris Karnovsky on the absorption of intravenously injected horseradish peroxidase (HRP) by proximal tubule epithelial cells of the mouse kidney, ¹ republished in this issue of the Journal of Histochemistry & Cytochemistry (JHC), is the most highly cited publication of any article to appear in JHC. At this writing, and according to the Google Scholar and Web of Science databases, the paper has been cited at least 7589 times. Among a recently compiled list of the most cited publications in the medical discipline of nephrology, the article ranks number 5. ² There are several reasons why this paper is so highly referenced, and these include the clarity in which the new peroxidase histochemical technique is described and the use of HRP as an in vivo macromolecular tracer at the ultrastructural level.

In his own retelling on the development of his peroxidase method, Karnovsky explained that the ultrastructural tracers available at the time (such as ferritin or colloidal gold) were much too large to simulate the passage of molecules smaller than albumin across endothelial cells. ³ Although smaller molecules and various enzymes including HRP (~40 kDa) had been used previously as in vivo tracers for light microscopy, the colorimetric stains or enzymatic reactions they generated were unsuitable for electron microscopy. Karnovsky therefore saw a need to identify an HRP enzyme substrate that would yield a stable, osmiophilic, electron-dense reaction product. He reasoned that diaminobenzidine tetrahydrochloride (DAB) might be a suitable electron donor that would be reduced by HRP in the presence of hydrogen peroxide to form an insoluble, electron-dense polymer. Together with Richard Graham, they defined optimal conditions for the development of the HRP-DAB reaction and its use as an ultrastructural tracer, which are presented in this paper.

The scientific premise of the article involves tracking the distribution of intravenously injected HRP in vivo as it makes its way from circulation into the proximal convoluted tubule of the mouse kidney. Kidney tissue is sampled 90 sec to 1 hr after injection and fixed and processed for peroxidase histochemistry using the newly developed technique. Including details on how to incubate HRP-laden tissue with hydrogen peroxide and DAB and then post-fix with osmium, the Materials and Methods section of the paper contains a few nuggets. First, there is a thorough description for the preparation of the combined formaldehyde-glutaraldehyde fixative (that soon became known as “Karnovsky’s fix”), which originally appeared only in abstract form (and is still one of the world’s most cited abstracts). ⁴ Another pro tip in the Methods section advised counterstaining ultrathin sections containing DAB reaction product with lead for clearer tissue visualization. (Counterstaining with uranyl acetate was not mentioned in the paper, and most electron microscopists routinely omit its use on DAB-developed tissue.)

The Results include eight spectacular electron micrographs of tissue fixed after intravenous injection of HRP showing peroxidase–DAB reaction product contained first within apical endocytic vesicles of kidney proximal tubular epithelial cells and, at slightly later intervals, lysosomes. Owing to the use of the dual, paraformaldehyde-glutaraldehyde fixative, tissue preservation is superb. The electron-dense, intracellular HRP reaction product is readily observed and confined to membrane-bound organelles. These results provided compelling evidence that the intravenously injected peroxidase retained enzymatic activity despite strong fixation conditions and could generate a clearly recognizable reaction product, proving the utility of this new ultrastructural tracer method.

Shortly after the publication of their original HRP-DAB paper, Graham and Karnovsky reported tests on mouse kidney glomerular permeability. ⁵ In this study, they sought to define the structural barriers within the glomerular capillary wall that restricted passage of plasma proteins. Intravenous injections of HRP and human myeloperoxidase (MPO, ~160 kDa) were used as visual surrogates for circulating macromolecules spanning the size of plasma albumin (~67 kDa). (Normally, albumin does not appreciably cross the glomerular filtration barrier and is retained in the circulation.) The tissue was preserved with Karnovsky’s fix at various intervals after injection and processed with DAB, which also serves as a substrate for MPO (and many other peroxidases). Unstained semi-thin sections (0.5–1 µm thick) examined by light microscopy clearly show the dark brown HRP reaction product within glomeruli and brush borders and endosomes of tubular epithelial cells. Electron microscopy of glomerular capillaries shows relatively rapid penetration of HRP through the glomerular basement membrane (GBM), between the epithelial slits of podocyte foot processes, into the urinary space of Bowman’s capsule and progressive accumulation within intracellular endosomes and lysosomes of proximal tubule epithelia. MPO, on the other hand, fails to cross into the glomerular filtrate and its reaction product accumulates at the level of the epithelial slits.

The findings from the glomerular permeability studies after intravenous injection of HRP and MPO led Graham and Karnovsky to propose that the primary filtration barrier to plasma macromolecules existed at the base of podocytes and specifically at the epithelial slits. ⁵ This was contrary to the views of Marilyn Farquhar and colleagues, whose work with non-enzymatic, particulate tracers favored the GBM as the principal glomerular filtration barrier. ⁶ Despite the many advantages of the HRP-DAB ultrastructural method, Farquhar pointed out certain limitations, including the potential diffusion of DAB reaction product beyond its site of production. Karnovsky and colleagues later reported that fixation conditions are especially important for glomerular permeability experiments and that, if the renal blood flow is interrupted, plasma proteins and injected tracers abnormally leak across the GBM.^7,8 Notably, with advanced understanding of the cell biology of the glomerular endothelial cells and podocytes, molecular composition of the GBM and epithelial slit diaphragms, and genetics of glomerular disease, the structural basis for glomerular permeability has continued to be debated over the ensuing decades. Although this matter is still not entirely resolved, most investigators agree that healthy glomerular endothelial cells, the GBM, and podocytes are all essential for normal filtration.

After the first description of the HRP-DAB method, another major advance in the use of HRP as an ultrastructural tracer came a few years later from Paul Nakane and Akira Kawaoi, who described a relatively straightforward method for conjugating IgG to HRP. ⁹ This oft-cited paper, which also appeared in JHC, described a procedure where reactive amino groups on HRP are first blocked with fluorodinitrobenzene to prevent self-coupling. Next, hydroxyl groups on HRP carbohydrate moieties are oxidized by periodate to yield aldehydes that can form Schiff bases with free amino groups on any protein, including immunoglobulins, and the bonds are stabilized with sodium borohydride. The periodate activation technique made it easy for any lab to conjugate their favorite antibodies to HRP. This advance ushered in a new era of immunoelectron microscopy using HRP–antibody conjugates and DAB, and also enabled the development of histopathology procedures that are commonly used today for immunolabeling cryostat and paraffin sections from clinical specimens.

Due to its relative ease, reproducibility, and practicality, the Graham and Karnovsky HRP-DAB method is used currently in a large assortment of biomedical research and clinical applications, with a variety of commercial kits widely available. In addition to immunohistochemical tools for light and electron microscopy, these include immunoassays such as ELISAs and immunoblot reagents where DAB reaction product, as well as those from its derivatives or other substrates, can be readily and accurately quantified. Nevertheless, the milestone HRP-DAB histochemical method, first published in JHC almost 57 years ago, remains a mainstay and extremely reliable light and electron microscopic technique for countless histopathology and research labs worldwide.