Western blot confirms type VII collagen content in retinal substrates.

<p>Western blot stained for type VII collagen. A 290 kDa band appeared in samples (directly) placed in reducing agent buffer (group/lane <b>A</b>). Omitting the use of reducing agents resulted in fading of the 290 kDa band (group/lane <b>B</b>). When samples were incubated in 30 units/ml collagenase, the 290 kDa band disappeared completely, while a 145 kDa band appeared (group/lane <b>C</b>).</p

Confocal microscopy image of retinal section.

<p>Type VII collagen-positive vesicles (LH7.2; green) around two cell nuclei (white arrowheads) in the inner plexiform layer (IPL). Note filamentous GFAP (polyclonal; red) labeling within the nerve fiber layer and around blood vessels. Within the lumen of blood vessels, autofluorescent erythrocytes (*) are seen. They can be discriminated from the corpus amylaceum (white arrow) in terms of fluorescence intensity, size and location. The inner limiting membrane (ILM) directly above the left blood vessel shows some focal positivity for type VII collagen labeling. A control section does not show any Col VII or GFAP labeling. GCL ganglion cell layer. (400x, inset 960x)</p

Immunohistochemical analysis at the vitreoretinal junction of a paraffin-embedded section.

<p>Anti-type VII collagen labeling with monoclonal LH7.2. This donor sample contains a lot of cells within the ganglion cell layer (GCL) that have Col VII-positive vesicles in their cytoplasm. Four corpora amylacea reside in the inner plexiform layer (IPL). (400x)</p

Retinal type VII collagen distribution.

<p>Immunohistochemical analysis at the vitreoretinal junction of a paraffin-embedded, serially sectioned (3–4 <i>μ</i>m) human donor eye evaluated by light microscopy. Anti-type VII collagen labeling with monoclonal LH7.2 (sections 1, 3 & 4) and periodic acid-Schiff (sections 2 & 5). Type VII collagen is present in astrocytic corpora amylacea. At least three corpora amylacea were sliced in this series, consecutively visualized in sections 1–2 (white arrows), sections 2–3 (black arrows) and sections 3–5 (black arrowheads). Corpora amylacea reside in the ganglion cell layer and inner plexiform layer (IPL). Type VII collagen is also present in small vesicles, clustered near large nuclei in the inner retina (white arrowhead). The vesicles reside outside the nucleus, and within the cytoplasm (sections 1, 2 & 3 compared). The cytoplasm of the type VII collagen yielding cell type seems more extensive than that of most other cells residing in the ganglion cell layer (GCL). Furthermore, their nuclei are larger and appear less dense in PAS (or hematoxylin) stains. The inner limiting membrane (ILM) does not label visibly for type VII collagen. Negative control section (NC) shows no labeling. (200 x)</p

Type VII collagen is visualized at the vitreoretinal interface by immuno-TEM (epon).

<p>Immuno-electron microscopic image of the inner limiting membrane of a human retina. (<b>A-C</b>) Immunogold-labeled polyclonal antibody directed against type VII collagen (black arrows) is found in the direct vicinity of vitreous fibrils (white arrows) at the vitreoretinal interface. Some gold labeling is also situated within the inner limiting membrane (ILM). (<b>A</b>) Anterior retina, showing sparse, clustered labeling. Vitreous collagen fibrils join the retina. Bar 1 μm. (<b>B</b>) The posterior retina has frequent gold labeling at a depth of 150–200 nm in the inner limiting membrane. Bar 500 nm. (<b>C</b>) Posterior retina, gold labeling for type VII collagen is mostly at the retinal side of the vitreous fibrils. Müller cell endfeet (M) remain unlabeled. vb vitreous body. (<b>D</b>) Negative control of posterior retina shows no labeling. Bar 1μm.</p

Type VII collagen is visualized within corpora amylacea (T8100).

<p>Immuno-electron microscopic image of the inner plexiform layer of a human retina. Immunogold-labeled polyclonal antibody directed against type VII collagen labels two corpora amylacea diffusely. Within the corpora amylacea, there is no evidence of fibrillar collagen or banding. Some background labeling is present. Bar 10 μm.</p

Colocalization of type VII collagen and GFAP within vesicle clusters and corpora amylacea.

<p>Image of the inner retina, paraffin section. Type VII collagen labeling (polyclonal; green) colocalizes with that of GFAP (monoclonal; red). The vesicle cluster around one nucleus (left white arrowhead) contains both Col-VII and GFAP-positive vesicles, while another cluster (right white arrowhead) has few Col VII-positive vesicles. Most vesicles contain both type VII collagen and GFAP and label yellowish, but others contain either Col-VII or GFAP. In this donor sample, no filamentous GFAP labeling is detected (by monoclonal antibodies) at the nerve fiber layer (NFL). Two corpora amylacea can be seen, both labeled dually (white arrows). ILM inner limiting membrane, GCL ganglion cell layer, IPL inner plexiform layer, vb vitreous body. (400x)</p