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Vol. 40, No. 11. pp. 1807-1808. 1992 Printed in US.A.

The Journal of Histochemistry and Cytochemistry Copyright 0 1992 by The Histochemical Society. Inc.

Letter to the Editor

Localization of Heparan Sulfate Proteoglycan in Basement Membranes -

In a recent issue of the Journal (1). we reexamined the localization of heparan sulfate proteoglycan (HSPG) in basement membranes in the hope of settling a controversy opposing investigators who used cationic probes and localized HSPG anionic groups mainly to the lamina lucida (3-5,9) and other researchers who used antibodies to HSPG core protein and localized it mainly to the lamina densa (6,7). We attributed the discrepancy to the use of glutaraldehyde fixation by cationic probe users and formaldehyde fixation by antibody users. When the tests for the anionic groups and core protein of HSPG were combined on the same sections, both were localized mainly in the lamina densa. However, the combined tests could be carried out after formaldehyde fixation but not after glutaraldehyde fixation. We therefore could not safely conclude that HSPG was localized in the lamina densa. The problem was recently settled when various tissues processed either by cryofixation and freeze substitution or by glutaraldehyde fixation, followed by freezing in Freon and freeze-substitution, showed nine different basement membranes composed of a lamina densa without lamina lucida, as illustrated in the mouse glomerular basement membrane (Figure 1). It wasconcluded, in accord with previous findings (2.8). that the lamina lucida was an artifact, at least in the nine tissues investigated. Moreover gold immunolabeling of basement membranes processed in this manner with antibodies against HSPG core protein localized this substance throughout the basement membrane (Figure 1). Counts of the number of gold particles per 0.1 pm on nine micrographs gave 0.83 2 0.33 in the proximal third (endothelial side), 1.18 * 0.33 in the central third (middle), and 0.79 * 0.25 in the distal third (visceral side) of this basement membrane. Such density was on the same order of magnitude as that reported in the basement membrane of the ciliary epithelium (1). Briefly, the controversy

is settled in an unexpected manner, since the HSPG is in the lamina densa which alone seems to constitute the basement membrane. E L. CHAW

s. Reccivcd for publication July 31, 1992:

Accepted August 3. 1992 (2L2769).

INOUE

C. F! L E B ~ N D Department of Anatomy McCill University Montreal. Quebec. Canada

Literature Cited 1. Chan FL, Inoue S, Leblond C P Localization of heparan sulfate proteoglycan in basement membrane by side chain staining with Cuprolinic blue as compared to core protein labeling with immunogold. J Histochem Cytochem 40:1559, 1992 2. Goldberg M, Escaig-Haye F: Is the lamina lucida of the basement membrane a fixation artefact? Eur J Cell Biol 42:365, 1986 3. Hay ED, Maier S Glycosaminoglycan synthesis by embryonic inductors: neural tube, notochord, and lens. J Cell Biol 62989, 1974

4. Kanwar YS, Farquhar M G Presence of heparan sulfate in the glomerular basement membrane. Proc Natl Acad Sci USA 761303. 1979 5. Kanwar YS. Farquhar MG: Anionic sites in the glomerular basement membrane. In vivo and in vitro localization to the lamina rarae by cationic probes. J Cell Biol 81:137, 1979 6. Laurie GW, Leblond CP, Inoue S, Martin GR. Chung A: Fine structure of the glomerular basement membrane and localization of five base-

Figure 1. Mouse glomerular basement membrane (BM) fixed by perfusion with 4% paraformaldehyde, followed by freezing in Freon, dehydration by freeze-substitution in acetone, and embedding in LR White. The section has been immunogold-labeledwith antibodies against the HSPG core protein. The gold particles are distributed over the entire glomerular basement membrane,which is filled with lamina densa material, so that no lamina lucida is present on either side. Ep. visceral epithelium; En, endothelium; Cap, lumen of fenestrated capillary. Original magnification x 91,800. Ear = 100 nm.

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LETTER TO THE EDITOR

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ment membrane components to the lamina densa and its extensions in both glomeruli and tubules of the rat kidney. Am J Anat 169:463, 1984

8. Reale E, Luciano L: The laminae rarae of the glomerular basement mem-

7. Palotie A, Peltonen L, FoidartJM, Rajaneimi H: Immunohistochemical localization of basement membrane components and interstitial colla&en types in preovulatory rat follicles. Col1 Relat Res 4:279, 1984

9. Tielstad U, Hayshi K.Toole BP: Epithelial collagens and glycosaminoglycans in the embryonic cornea. Macromolecular order and morphogenesis in the basement membrane. J Cell Biol 62:815, 1974

brane. Hereditary nephritis. Contrib Nephrol 80:32, 1990

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Localization of heparan sulfate proteoglycan in basement membranes.

0022-1554/921$3.30 Vol. 40, No. 11. pp. 1807-1808. 1992 Printed in US.A. The Journal of Histochemistry and Cytochemistry Copyright 0 1992 by The His...
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