Journal: PLoS ONE

Article Title: The Bi-Functional Organization of Human Basement Membranes

doi: 10.1371/journal.pone.0067660

Figure Lengend Snippet: The retinal surface (R) of the ILM and the endothelial (En) and epithelial (Ep) sides of DM and LC were stained with a polyclonal (red, LNp; A, C, D) and a monoclonal antibody to laminin (red, LNm, B), whereas the vitreal (V) or stromal side (St) of the BMs were labeled with an antibody to the 7S domain of collagen IV α3/4/5 (green; A-C, E). The asymmetry of BMs was also detected by single and double labeling of crossections of an isolated ILM (F-H) and an ILM in situ (I). The TEM micrographs in panel (K, L) show crossections of isolated ILMs stained for 7S collagen IV α3/4/5 (K) and laminin (L). The dark label shows the localization of 7S collagen IV on the vitreal side (K) and laminin on retinal side of the ILM (L). An asymmetric distribution for laminin and collagen 7S was also detected for the Descemet’s membranes (M, N) and the lens capsule (O, P). The sections were stained for laminin (red; M-P) and collagen IV 7S (green; N, P). Scale Bars: A-E: 100 µm; F-I and M-P: 10 µm; K, L, I: 1 µm.

Article Snippet: The blots were incubated with a polyclonal anti-collagen IV antibody (Rockland) followed by alkaline-phosphatase-conjugated goat anti-rabbit (1∶2000; Jackson ImmunoResearch, West grove, PA).

Techniques: Staining, Labeling, Isolation, In Situ

Journal: PLoS ONE

Article Title: The Bi-Functional Organization of Human Basement Membranes

doi: 10.1371/journal.pone.0067660

Figure Lengend Snippet: The domain specificity was determined by Western blotting (lanes 1–6, panel A) and immunoprecipitation (lanes 7, 8, panel A) using adult human lens capsules as sample. The western blot of lanes 1 and 2, stained with a polyclonal antiserum to human collagen IV, shows the complex peptide banding pattern of intact collagen IV from human lens capsules age 24 (lane 1) and age 65 (lane 2). Staining of the 65 year-old lens capsule sample with the J3-2 Mab (lane 3) shows a similar, but not entirely identical banding pattern. Red bars mark corresponding bands. The non-collagenous (NC1 and 7S) domains of collagen IV were detected in western blots after digestion of lens capsules with collagenase: western blots with the soluble supernatant from digested lens capsule samples, stained with polyclonal collagen IV antiserum, shows the NC-domains of collagen IV as a long smear at 300 kD and a ladder of lower molecular weight peptides (lane 4, panel A). The NC1 domain of α3 collagen IV appears as a sharp band below 49 kD (lane 5, red bar; panel A) as shown by staining with a monoclonal antibody specific to the collagen IV α3 NC1 domain. The high molecular weight smear stained by Mab J3-2 correspond to the variably crosslinked 7S domain (Lane 6, panel A). The smear is due to the glycosylation of the 7S domain , . For immunoprecipitation, the collagenase-digest of lens capsule was incubated with Mab J3-2 or anti-laminin as a control followed by anti-mouse IgM or anti-rabbit IgG agarose. The beads were washed and the bound peptide released by boiling in high molar urea/SDS sample buffer. The released peptides were separated by SDS PAGE and western blotted. The immuno-precipitated peptides were visualized in the blot by a polyclonal anti-collagen IV antibody/alkaline phosphatase-conjugated secondary antibody. For the J3-2 pull-down, a smear of approximately 300 kD was detected (lane 7, panel A), similar to the smear detected by Western blotting (lane 6). A control pull-down with anti-laminin is shown in lane 8. The blots shows that Mab J3-2 i) recognizes human collagen IV, that it ii) recognizes an NC domain of collagen IV and that iii) the molecular weight of the detected peptides are different from the molecular weight of the NC1 domain but identical to the molecular weight of the 7S domain of collagen IV from glomerular BM and lens capsule. To determine the chain-specificity of the antibody, sections of adult human retina were stained with Mab J3-2 (B), to the NC1 domain of collagen IV α3 (C), to the NC1 domain of collagen IV α1 (D) and to the NC1 domain of collagen α5 (E). A3, α4 (not shown) and α5 collagen IV are very prominent in the ILM but sparse in the BMs of the retinal blood vessels (C, E), whereas α1/2 was very sparse in the ILM but abundant in the blood vessels (D). The staining with Mab J3-2 (A) resembles most closely the staining of collagen α3 α4 and α5, and it is very different from the distribution of collagen IV α1/α2. Scale Bar: 50 µm.

Article Snippet: The blots were incubated with a polyclonal anti-collagen IV antibody (Rockland) followed by alkaline-phosphatase-conjugated goat anti-rabbit (1∶2000; Jackson ImmunoResearch, West grove, PA).

Techniques: Western Blot, Immunoprecipitation, Capsules, Staining, Molecular Weight, High Molecular Weight, Glycoproteomics, Incubation, Control, SDS Page

Journal: PLoS ONE

Article Title: The Bi-Functional Organization of Human Basement Membranes

doi: 10.1371/journal.pone.0067660

Figure Lengend Snippet: Staining with polyclonal antibodies resulted in a uniform and even labeling of the retinal (R) and vitreal (V) side of the ILM. In contrast, staining of ILM with antibodies specific to the 7S (B) or the NC1 domain (C) of collagen IV alpha3/4/5 resulted in the selective labeling of the vitreal (B) or the retinal side (C). The distribution of laminin in the ILM (D) is very similar to that of collagen IV NC1 at the epithelial side. The ILM sample in panel C and D was double-labeled; the NC1 domain was labeled with a Cy3 (red; C) secondary antibody, whereas laminin was detected with a Cy2 (green; D) secondary antibody. Scale Bar: 100 µm.

Article Snippet: The blots were incubated with a polyclonal anti-collagen IV antibody (Rockland) followed by alkaline-phosphatase-conjugated goat anti-rabbit (1∶2000; Jackson ImmunoResearch, West grove, PA).

Techniques: Staining, Labeling