Journal: Investigative ophthalmology & visual science

Article Title: Coordinated Intervention of Microglial and Müller Cells in Light-Induced Retinal Degeneration.

doi: 10.1167/iovs.61.3.47

Figure Lengend Snippet: FIGURE 3. The rings of cone degeneration are occupied by rosettes of Müller cells. Magnifications of the same regions of representative retinas immunodetected for L/M opsin (left column), GFAP (center column), or a merged image (right column) to show the rings of L/M-cone degeneration at 1 (first row), 2 (second row), and 3 (third row) months ALE. The rings devoid of L/M-cones are occupied by radial processes of Müller cells in the form of rosettes.

Article Snippet: Tissue fixation was performed by transcardial perfusion of the rats, first with saline and then with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), following standard protocols of our laboratory.3,56,57,63 The retinas were then dissected as whole-mounts and double immunodetection was performed following previously described methods.13,23,56,57,64 Primary antibodies were used to immunodetect different retinal cell populations: (1) microglial cells (rabbit anti-Iba1; Ionized Calcium-Binding Adapter Molecule 1; AB_839504; 1:1000, 019-19741; Wako Chemicals, Neuss, Germany), (2) astrocytes and Müller cells (goat anti-GFAP; Glial Fibrillary Acidic Protein; AB_641021; 1:250, C-19, sc-6170; Santa Cruz Biotechnology, Heidelberg, Germany, or Guinea Pig antiGFAP; AB_10641162; 173-004 Synaptic System, Goettingen, Germany), (3) Müller cells (goat anti-vimentin; AB_793998; 1:100, C-20, sc-7557; Santa Cruz Biotechnology), (4) L/Mcones (rabbit anti-L/M-opsin; AB_177456; 1:1200; ChemiconMillipore Iberica, Madrid, Spain), and (5) S-cones (goat anti-S-opsin; N-20; anti-OPN1SW; AB_2158332; 1:1000; Santa Cruz Biotechnologies).

Techniques:

Journal: Investigative ophthalmology & visual science

Article Title: Coordinated Intervention of Microglial and Müller Cells in Light-Induced Retinal Degeneration.

doi: 10.1167/iovs.61.3.47

Figure Lengend Snippet: FIGURE 5. Microglial and Müller cell involvement during cone S degeneration. Magnifications of the same regions from the superior retina of representative animals processed (A-A′′) 1, (B-B′′) 2, and (C-C′′) 3 months ALE and immunoreacted for S-opsin and Iba-1 (left column), GFAP (purple, central column), or merged images (right column). One month ALE, GFAP-labeled processes of Müller cells with a radial distribution were seen inside the (A-A′′) rings and activated microglial cells were usually seen in the borders of the rings, but there were also some in the center of some rings. Two months ALE, the rings merged, and GFAP immunoreactivity decreased at the center of the rings, whereas at their periphery, the GFAP-positive processes lost in part their radial distribution (B′). Three months ALE, the areas completely devoid of cones (left part of C-C′′) were filled by activated microglia that had resumed a mosaic distribution (C, C′′) and GFAP-positive processes that were radially oriented only at the boundaries of the cone degenerated area (C′, C′′). Arrows point to activated microglial cells inside of a ring of cone degeneration.

Article Snippet: Tissue fixation was performed by transcardial perfusion of the rats, first with saline and then with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), following standard protocols of our laboratory.3,56,57,63 The retinas were then dissected as whole-mounts and double immunodetection was performed following previously described methods.13,23,56,57,64 Primary antibodies were used to immunodetect different retinal cell populations: (1) microglial cells (rabbit anti-Iba1; Ionized Calcium-Binding Adapter Molecule 1; AB_839504; 1:1000, 019-19741; Wako Chemicals, Neuss, Germany), (2) astrocytes and Müller cells (goat anti-GFAP; Glial Fibrillary Acidic Protein; AB_641021; 1:250, C-19, sc-6170; Santa Cruz Biotechnology, Heidelberg, Germany, or Guinea Pig antiGFAP; AB_10641162; 173-004 Synaptic System, Goettingen, Germany), (3) Müller cells (goat anti-vimentin; AB_793998; 1:100, C-20, sc-7557; Santa Cruz Biotechnology), (4) L/Mcones (rabbit anti-L/M-opsin; AB_177456; 1:1200; ChemiconMillipore Iberica, Madrid, Spain), and (5) S-cones (goat anti-S-opsin; N-20; anti-OPN1SW; AB_2158332; 1:1000; Santa Cruz Biotechnologies).

Techniques: Labeling

Journal: Investigative ophthalmology & visual science

Article Title: Coordinated Intervention of Microglial and Müller Cells in Light-Induced Retinal Degeneration.

doi: 10.1167/iovs.61.3.47

Figure Lengend Snippet: FIGURE 6. GFAP-positive radial processes within the rings of cone degeneration correspond to the external processes of Müller cells. Confocal photomicrophotographs of representative rings of cone degeneration in retinal whole mounts 2 (A-A′′) or 3 (B-B′′) months ALE immunoreacted for Vimentin (A, B, A′′, B′′) and GFAP (A′, B′, A′′, B′′, C–F). The GFAP radial immunoreactive processes observed in the center of the rings, 2 (first row) or 3 (second row) months ALE are also Vimentin (A′′, B′′) positive, and thus correspond to the outer processes of Müller cells. Confocal three-dimensional reconstructions of the GFAP immunoreactivity in a ring of cone degeneration using different tilting degrees (C–E), to show GFAP immunoreactivity through the retina. The inner and outer retina have been indicated in the z axis. There are GFAP-labeled Müller cell processes in the inner retina that run vertically and become horizontal and radially oriented in the

Article Snippet: Tissue fixation was performed by transcardial perfusion of the rats, first with saline and then with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), following standard protocols of our laboratory.3,56,57,63 The retinas were then dissected as whole-mounts and double immunodetection was performed following previously described methods.13,23,56,57,64 Primary antibodies were used to immunodetect different retinal cell populations: (1) microglial cells (rabbit anti-Iba1; Ionized Calcium-Binding Adapter Molecule 1; AB_839504; 1:1000, 019-19741; Wako Chemicals, Neuss, Germany), (2) astrocytes and Müller cells (goat anti-GFAP; Glial Fibrillary Acidic Protein; AB_641021; 1:250, C-19, sc-6170; Santa Cruz Biotechnology, Heidelberg, Germany, or Guinea Pig antiGFAP; AB_10641162; 173-004 Synaptic System, Goettingen, Germany), (3) Müller cells (goat anti-vimentin; AB_793998; 1:100, C-20, sc-7557; Santa Cruz Biotechnology), (4) L/Mcones (rabbit anti-L/M-opsin; AB_177456; 1:1200; ChemiconMillipore Iberica, Madrid, Spain), and (5) S-cones (goat anti-S-opsin; N-20; anti-OPN1SW; AB_2158332; 1:1000; Santa Cruz Biotechnologies).

Techniques: Labeling