embedding [28]. For IEM analyses, specimens were adsorbed to a carbon-coated grid, blocked for 10 min in 1% BSA, immunola- beled with the first detection antibody using anti-Ab monoclonal antibody 4G8 (Signet Laboratories, Covance, Dedham, MA), then incubated with rabbit anti-mouse bridging antibody followed by incubation with 10 nm immunogold-conjugated Protein A (Jan Slot, Utrecht, Netherlands). Grids were fixed briefly in 1% glutaraldehyde, floated on 4 drops of 0.02M glycine/PBS to quench free aldehyde groups, then immunolabeled with the second detection antibody using a rabbit anti-aB-crystallin polyclonal antibody (Dr. Jack Liang, Brigham & Women’s Hospital, Boston, MA, USA) or normal rabbit serum control. Specimens were then exposed to 15 nm immunogold-conjugated Protein A (Jan Slot, Utrecht, Netherlands), negatively stained with uranyl acetate, and examined on a JEOL 1200EX transmission electron microscope at the Electron Microscope Facility, Harvard Medical School. All immunohistochemical analyses included specificity controls in which identically processed specimens were incubated with detection antibody that was immunodepleted by pre-absorption with synthetic antigen (human Ab42, Keck Laboratory, Yale University, New Haven, CT) as previously described[28]. Tryptic Digest Tandem Mass Spectrometry Protein Sequencing Lens specimens were homogenized in 70% formic acid, ultra- centrifuged at 100,0006g for 1 h at 4uC, and fractions separately retained for biochemical analysis. Supernatant was concentrated by nitrogen stream, neutralized with NaOH, and diluted 50-fold in TBS containing 10 mg anti-Ab monoclonal antibody 6E10 (Signet Laboratories, Covance, Dedham, MA). Immunocomplexes were precipitated with magnetic beads covalently coupled to sheep anti- mouse antibody (Invitrogen, Carlsbad, CA), separated from unbound material by exposure to a magnetic field, and released by acidification in 100 mM glycine at pH 3. Samples were then subjected to reverse phase HPLC on a ZORBAX SB-C8 column (Agilent, Santa Clara, CA) and eluted under isocratic conditions (0.1% TFA; 30.5% acetonitrile) at 1 ml/min at 80uC. Elution times were determined by comparison to chromatographic profiles of synthetic human Ab (Keck Laboratory, Yale University, New Haven, CT). Isolated fractions were dried by vacuum centrifuga- tion, digested with trypsin, and fractionated by reverse phase HPLC. Post-digest eluates were subjected to electrospray ioniza- tion and ESI-tandem MS/MS LCQ-DECA ion-trap mass spectrometry (ThermoFinnigan, San Jose, CA) at the Taplin Mass Spectrometry Facility, Harvard Medical School. Peptide sequenc- es were identified by computer-assisted search program (Sequest, Thermo Fisher Scientific, Waltham, MA). Ab Fluoro-ELISA and Ab Immunoblotting Analytical quantification of human Ab40 and human Ab42 was accomplished using two commercially available human Ab specific ELISA kits (BetaMark, Covance, Dedham, MA; BioSource, Invitrogen, San Diego, CA). For experiments utilizing SDS extracted tissue, we used a commercially available human Ab ELISA kit (BetaMark, Covance, Dedham, MA) that is resistant to interference from detergent. This ELISA assay system is compatible with tissue extracts containing SDS with minimal loss in sensitivity. Samples containing SDS were diluted to a final detergent concentration of less than 0.1%. For experiments utilizing formic acid extracted tissue, we used a second commercially available Ab ELISA kit (BioSource, Invitrogen, San Diego, CA). For immunoblotting analysis, lens subcellular fractions were normalized for protein concentration and analyzed Ab in Lens in Down Syndrome as previously described [28]. Blots were probed with human Ab- specific monoclonal antibody WO2 (Genetics Company, Zurich, Switzerland) directed against human Ab4–10 or monoclonal antibody 6E10 (Signet Laboratories, Covance, Dedham, MA) directed against human Ab3–8. Quasi-Elastic Light Scattering Analysis of Human Lens Protein Suspensions Quasi-elastic light scattering (QLS) experiments were conducted using a custom laboratory instrument (Neuroptix Corporation, Acton, MA) that delivers linearly polarized continuous wave near- infrared laser light (l = 780 nm; focused beam diameter, 30– 35 mm; power output = 0.3 mW). The instrument incorporates a rigidly positioned pinhole mirror (aperture diameter = 100 mm) for selective detection of backscattered photons at a fixed scattering angle (h = 120 degrees). Signal detection and analysis of backscat- tered light intensity was accomplished with an avalanche photodiode, integrated 256-channel multi-tau digital autocorre- lator (t = 480 ns), and custom software analysis package. Analyses involving aqueous suspensions of water-soluble lens proteins were conducted in cylindrical optical glass specimen vials positioned in a rigid holder mounted to the instrument. Three series of five independent measurements (each 1.3 seconds in duration) were performed for a total of fifteen measurements comprising each averaged test point. To investigate the interaction of Ab peptides with other cytosolic lens proteins, a membrane-free water-soluble protein extract was prepared from freshly dissected human lenses obtained from postmortem donor eyes (National Disease Registry Interchange, Philadelphia; PA; Florida Lion’s Eye Bank, Miami, FL). Dissected lenses were mechanically homogenized, ultracen- trifuged at 100,0006g for 1 h at 4uC, and diluted to 1 mg/ml in phosphate buffered saline, pH 7.20. This aqueous lens protein extract is predominantly composed of water-soluble proteins in the cytoplasm of lens fiber cells. Purified synthetic human Ab42 or Ab40 (Keck Laboratories, Yale University, New Haven, CT) was added to freshly prepared human lens protein extract and incubated in siliconized Eppendorf tubes in a humidified 37uC 5% CO2-balanced tissue culture chamber prior to light scattering analysis. All samples were incubated in the dark to reduce photodynamic effects. For microscopic visualization, aliquots of the incubated samples were placed on a microscope slide as wet- mount preparations and visualized by conventional photomicros- copy under brightfield and cross-polarized illumination. Immuno- gold electron microscopic analysis of incubated material was conducted as detailed above. Results Supranuclear Cataract Pathology in Down Syndrome Lenses Slit lamp examination of subjects with karyotype-confirmed DS revealed classical cerulean ‘‘blue dot’’ cataracts (Fig 1A) with a characteristic subequatorial supranuclear distribution (Fig. 1B,C) and corresponding increased localized static light scattering (Fig. 1D). The anatomical localization of the diffuse opacities observed by indirect ophthalmoscopy (Fig. 1A) correspond to a distinctly annular supranuclear distribution as revealed by retro- illumination (Fig. 1B,C) and confirmed by scattering densitometry (Fig. 1D) of the same lens. The lens from a 2-year-old male with DS demonstrated grossly normal transparency without evidence of clinically detectable lenticular pathology (Fig. 1E). By contrast, the DS supranuclear cataract phenotype was identified in all adult DS patients clinically examined and in all postmortem lens specimens from adult DS donors. Lenses from mature DS subjects PLoS ONE | www.plosone.org 3 May 2010 | Volume 5 | Issue 5 | e10659