Journal: Experimental eye research

Article Title: Decreased endostatin in db/db retinas is associated with optic disc intravitreal vascularization.

doi: 10.1016/j.exer.2021.108801

Figure Lengend Snippet: Fig. 1. Endostatin staining and antibody specificity in mouse retina. (A) Immunofluorescence with a rabbit polyclonal anti-endostatin antibody in mice retina showed staining in the ganglion cell layer, outer plexiform layer, and in the outer segments of photoreceptors (left panel). Blocking peptide assay (Blocking PA) using recombinant mouse endostatin was performed to assess antibody specificity. Anti-endostatin antibody neutralization was complete and non-specific signal was observed in the retina (middle panel). Negative control was done by omission of the primary antibody (right panel). (B) FcR blocking assay confirmed the specificity of endostatin staining (left panel) when compared to retinas incubated with Mouse Seroblock FcR (middle panel). Negative control was done by omission of the primary antibody (right panel). (C) Isotype control assay using an anti-collagen I antibody from the same isotype and host as the anti-endostatin antibody was performed. Collagen I is absent from retina and very abundant in cornea. In comparison with the anti-endostatin antibody retinal signal (left panel), non-signal was observed in the retina when using anti-collagen I antibody (middle panel). Positive control for collagen I was performed in the cornea of the same eye in a consecutive section (right panel). Nuclei were counterstained with Hoechst. GCL: ganglion cell layer; INL: inner nuclear layer; ONL: outer nuclear layer; Co: cornea. Scale bars: 37.03 μm.

Article Snippet: For blocking peptide assay, the neutralization of the primary anti-endostatin antibody was done in a previous incubation step of 1 h before immunohistochemistry by adding 10-times excess of recombinant mouse endostatin (R&D Systems, MN, Minneapolis), in contrast to the control incubated with the anti-endostatin antibody alone.

Techniques: Staining, Immunofluorescence, Blocking Assay, Recombinant, Neutralization, Negative Control, Incubation, Control, Comparison, Positive Control

Journal: Experimental eye research

Article Title: Decreased endostatin in db/db retinas is associated with optic disc intravitreal vascularization.

doi: 10.1016/j.exer.2021.108801

Figure Lengend Snippet: Fig. 3. Endostatin in mouse neuroretina. (A) Immunostaining with anti-endostatin antibody revealed that endostatin is accumulated in the inner part of neuroretina, however, it was not observable at the optic disc (B) No endostatin signal was detected in blood vessel basement membranes or in the internal limiting membrane. Nuclei were counterstained with Hoechst. GCL: ganglion cell layer; INL: inner nuclear layer; ONL: outer nuclear layer; BBM: blood vessel basement membrane; ILM: internal limiting membrane. Scale bars: 43.48 μm (A); 33.33 μm (B).

Article Snippet: For blocking peptide assay, the neutralization of the primary anti-endostatin antibody was done in a previous incubation step of 1 h before immunohistochemistry by adding 10-times excess of recombinant mouse endostatin (R&D Systems, MN, Minneapolis), in contrast to the control incubated with the anti-endostatin antibody alone.

Techniques: Immunostaining, Membrane

Journal: Experimental eye research

Article Title: Decreased endostatin in db/db retinas is associated with optic disc intravitreal vascularization.

doi: 10.1016/j.exer.2021.108801

Figure Lengend Snippet: Fig. 2. Western blot analysis of mouse retinal endostatin. Retinal endostatin shows a main band between 15 and 20 kD (lane 1), very close to the 21 kDa band from the recombinant endostatin (lane 2), confirming the antibody specificity for cleaved endostatin. α-tubulin was used as a loading control.

Article Snippet: For blocking peptide assay, the neutralization of the primary anti-endostatin antibody was done in a previous incubation step of 1 h before immunohistochemistry by adding 10-times excess of recombinant mouse endostatin (R&D Systems, MN, Minneapolis), in contrast to the control incubated with the anti-endostatin antibody alone.

Techniques: Western Blot, Recombinant, Control

Journal: Experimental eye research

Article Title: Decreased endostatin in db/db retinas is associated with optic disc intravitreal vascularization.

doi: 10.1016/j.exer.2021.108801

Figure Lengend Snippet: Fig. 4. Endostatin is accumulated in bipolar cells of mouse retina. Double immunostaining with anti-PKCα antibody confirmed that endostatin was accumulated mainly in bipolar cells. Endostatin was found in their axon terminals (arrowheads), but not in their axons themselves (arrows) (A) and in the perikaryon and dendrites (arrowheads) (B). Nuclei were counterstained with Hoechst. GCL: ganglion cell layer; IPL: inner plexiform layer; INL: inner nuclear layer; OPL: outer plexiform layer; ONL: outer nuclear layer. Scale bar: 25.42 μm; 9.41 μm (A); 10.17 μm (B).

Article Snippet: For blocking peptide assay, the neutralization of the primary anti-endostatin antibody was done in a previous incubation step of 1 h before immunohistochemistry by adding 10-times excess of recombinant mouse endostatin (R&D Systems, MN, Minneapolis), in contrast to the control incubated with the anti-endostatin antibody alone.

Techniques: Double Immunostaining

Journal: Experimental eye research

Article Title: Decreased endostatin in db/db retinas is associated with optic disc intravitreal vascularization.

doi: 10.1016/j.exer.2021.108801

Figure Lengend Snippet: Fig. 5. Endostatin is present in cone photoreceptors of mice retina. Double immunostaining with anti-cone arrestin antibody showed endostatin in the inner seg ments of cone photoreceptors (arrows). Cone synaptic terminals did not show endostatin expression (arrowheads). Nuclei were counterstained with Hoechst. GCL: ganglion cell layer; INL: inner nuclear layer; OPL: outer plexiform layer; ONL: outer nuclear layer; IS: inner segments of photoreceptors. Scale bar: 25.88 μm; 17.25 μm (inset).

Article Snippet: For blocking peptide assay, the neutralization of the primary anti-endostatin antibody was done in a previous incubation step of 1 h before immunohistochemistry by adding 10-times excess of recombinant mouse endostatin (R&D Systems, MN, Minneapolis), in contrast to the control incubated with the anti-endostatin antibody alone.

Techniques: Double Immunostaining, Expressing

Journal: Experimental eye research

Article Title: Decreased endostatin in db/db retinas is associated with optic disc intravitreal vascularization.

doi: 10.1016/j.exer.2021.108801

Figure Lengend Snippet: Fig. 6. Human optic disc was practically devoid of endostatin. Double immunostainig with anti- endostatin and anti-collagen IV antibodies showed a markedly endostatin accumulation in the neuroretina, whereas as happens in mouse retina, endostatin was unobservable in the optic disc. The figure is a mosaic composite obtained from different captures at 63X in the laser scan ning confocal microscope. OD: Optic disc; ON: Optic nerve; CR: Central retinal artery and vein; R: Retina. Scale bar: 474.92 μm; 226.15 μm (inset).

Article Snippet: For blocking peptide assay, the neutralization of the primary anti-endostatin antibody was done in a previous incubation step of 1 h before immunohistochemistry by adding 10-times excess of recombinant mouse endostatin (R&D Systems, MN, Minneapolis), in contrast to the control incubated with the anti-endostatin antibody alone.

Techniques: Microscopy

Journal: Experimental eye research

Article Title: Decreased endostatin in db/db retinas is associated with optic disc intravitreal vascularization.

doi: 10.1016/j.exer.2021.108801

Figure Lengend Snippet: Fig. 7. Comparison between human and mouse endostatin distribution in neuro retina. Immunolabelling with anti- endostatin antibody in human and mouse retinas showed a similar, although with some differences, endostatin distribution in both species, mainly located at bipolar cells and photoreceptors. (A) Human bipolar ter minal axons (arrows) showed lower expres sion of endostatin compared to mouse retina. (B) Bipolar cell perikaryal were stained with endostatin in both human and mice retina with a similar intensity. (C) In contrast with mice, where only scarce cones (arrowheads) showed endostatin accumula tion in their inner segments, human retina showed a higher signal of endostatin at photoreceptors, suggesting that both cone and photoreceptors accumulated endostatin. The same anti-endostatin antibody was used on both human and mice retinal sections. GCL: ganglion cell layer; IPL: inner plexi form layer; INL, inner nuclear layer; OPL: outer plexiform layer; ONL: outer nuclear layer; IS: inner segments of photoreceptors. Scale bars: 38.3 μm; 10.64 μm (A, B, C).

Article Snippet: For blocking peptide assay, the neutralization of the primary anti-endostatin antibody was done in a previous incubation step of 1 h before immunohistochemistry by adding 10-times excess of recombinant mouse endostatin (R&D Systems, MN, Minneapolis), in contrast to the control incubated with the anti-endostatin antibody alone.

Techniques: Comparison, Staining

Journal: Experimental eye research

Article Title: Decreased endostatin in db/db retinas is associated with optic disc intravitreal vascularization.

doi: 10.1016/j.exer.2021.108801

Figure Lengend Snippet: Fig. 8. Endostatin is decreased in db/db mouse retinas. (A) Western blot analysis revealed that endostatin protein expression was 0.5-fold decreased in 12 weeks-old diabetic mouse retinas, especially in the ganglion cell layer. Statistical analysis showed significant differences in endostatin band intensity between db/+ and db/db mice (1.275 ± 0.126 arbitrary units in db/+ mice vs. 0.753 ± 0.055 arbitrary units in db/db mice; p = 0.019; n = 3). α-tubulin was used as a loading control (B) Immunofluorescence representative images showing decreased endostatin expression in db/db retinas. Nuclei were coun terstained with Hoechst. OD: Optic disc; CR: Central retinal vessel; R: Retina. Scale bar: 22.22 μm.

Article Snippet: For blocking peptide assay, the neutralization of the primary anti-endostatin antibody was done in a previous incubation step of 1 h before immunohistochemistry by adding 10-times excess of recombinant mouse endostatin (R&D Systems, MN, Minneapolis), in contrast to the control incubated with the anti-endostatin antibody alone.

Techniques: Western Blot, Expressing, Control, Immunofluorescence