Tissues were fixed in 10% buffered formaldehyde and processed for light microscopy (27 (link)). For immunohistochemistry, paraffin sections were warmed to 50°C for 30 min, dewaxed in xylene, and rehydrated through decreasing concentrations of ethanol. Endogenous peroxidase activity was blocked by a 15-min preincubation with methanolic H2O2 (0.5% vol/vol). After washing in TBS, pH 7.5, the sections were incubated with normal goat serum (1:200) for 30 min, washed extensively with TBS, and incubated with the rabbit polyclonal LF-113 anti-decorin or the LF-106 antibiglycan antisera (16 ) at 1:1,000 dilution for 18 h at 4°C. The LF-106 is directed against a murine biglycan synthetic peptide, amino acids 50–64, conjugated to horseshoe crab hemocyanin (16 ). An HRP-conjugated goat anti–rabbit IgG (Sigma Chemical Co.) was applied at 1:200 dilution for 45 min. A 3-amino-ethyl carbazole substrate kit (AEC; Vector Laboratories Inc., Burlingame, CA) was used to visualize the specific peroxidase activity. The sections were washed in water, counterstained with 0.2% methylene blue, and mounted with Gel/Mount aqueous medium (Biomeda Co., Foster City, CA) before photography. For EM, small portions of skin, tail tendon, or cornea were fixed in 3% glutaraldehyde, 25 mM sodium acetate buffer, pH 5.7, containing 0.3 M MgCl2 and 0.05% cuprolinic blue (53 (link)). The unosmicated tissues were rinsed three times in buffer containing MgCl2, en bloc stained with 1% sodium tungstate, dehydrated in graded alcohols, and embedded in Spurr's epoxy resin. Thin-sections were observed with a transmission electron microscope 100B (JEOL USA, Peabody, MA), with or without further staining with uranyl acetate (27 (link)) or sodium tungstate (53 (link)). For quantitative studies, collagen fibril diameters were measured on photographic prints with a calibrated final magnification of 90,000. Several hundred micrographs were taken from the skin, tendon, and cornea of five homozygous, two heterozygous and three wildtype animals. A total of 2,803 collagen profiles from wild-type, heterozygous, and homozygous skin was measured, and histograms were generated. For scanning transmission EM (STEM) analysis, 4-mm2 pieces of skin were finely minced, suspended in 1 ml Tris-buffered (pH 7.4) saline supplemented with 50 mM EDTA and 100 mM sucrose, and subjected to mild disruption in a hand-held Dounce homogenizer. The supernatants were then sampled for EM. Fibrils were adsorbed to 400-mesh carbonfilmed grids, washed with ultrapure water, and air dried. The unstained fibrils were examined by STEM and mass mapping procedures (21 (link), 22 (link), 23 (link)).