anti il 1b goat polyclonal antibody  (R&D Systems)

Journal: The American journal of pathology

Article Title: Choroidal Involution Is Associated with a Progressive Degeneration of the Outer Retinal Function in a Model of Retinopathy of Prematurity: Early Role for IL-1β.

doi: 10.1016/j.ajpath.2016.08.004

Figure Lengend Snippet: Figure 1 IL-1b is immediately and markedly elevated in the RPE/choroid (Ch) complex from rats exposed to the oxygen-induced retinopathy (OIR) model. A: Q58 Graph showing the quantitative PCR analysis on RPE/choroid from animals exposed to cycling oxygen (50% and 10%) or their nor- moxic controls (NOR; set as 1). During early in- duction of retinopathy (at P10), proinflammatory IL-1b, IL-6, and tumor necrosis factor (TNF)-a were up-regulated (3.17-, 2.15-, and 1.80-fold, respectively). Immediately after completing cycling oxygen exposure (at P14), IL-1b increased to 6.3-fold in the RPE/choroid. In comparison, IL- 6 and TNF-a were moderately increased (2.65- and 3.30-fold, respectively). Fold changes were normalized to 18S as internal control. B: Western blot of RPE/choroid complex from normoxic con- trols, OIR rats, and/or OIR rats treated with IL-1 receptor antagonist (IL-1ra; Kineret). IL-1b (17 kDa) protein level was marked increased in OIR rats but significantly reverted with IL-1ra admin- istration. b-Actin protein (42 kDa) was used as internal control. C: Immunohistochemistry revealed IL-1b (green arrows) was colocalized with the RPE layer (red arrow) of OIR animals. D: Immunochemistry indicated specific colocalization between NACHT, LRR, and PYD domains- containing protein 3 (NLRP-3) (green arrows), a component of inflammasome that produces IL-1b, and retinal pigment epithelium-specific 65-kDa protein (RPE65) in P14 OIR animals. E: Immuno- chemistry indicated specific localization of cas- pase 1, the active enzyme that produces mature IL-1b, in the RPE of P14 OIR animals (red arrows). Data are expressed as means SEM Q59 (-). n Z 3 to 4 experiments, and representative images from 4 experiments are shown (-). *P < 0.05, **P < 0.01. Scale bars: 10 mm (C); 50 mm (D and E). P, postnatal day Q60 .

Article Snippet: Because both control and treated eyes were embedded in the same mode and stained with the same procedure, the differences induced by artifacts are minimized. ajp.amjpathol.org - The American Journal of Pathology 496 18 October 2016 5:27 pm EO: AJP16_0032 Q36 Q37 1⁄2T2 Q38 Q39 1⁄2F1 1⁄2F2 Q7 Choroidal Involution and Its Long-Term Impact on Retinal Degeneration 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 Western Blot Protein extracts (40 mg) were loaded, separated by voltage potential, and transferred to polyscreen polyvinylidene difluoride transfer membrane (PerkinElmer), as previously reported.23 Respective proteins were revealed by goat antie IL-1b polyclonal antibody (AF-501-NA, 1:500; R&D Systems) or mouse antieb-actin monoclonal antibody (NB600-501, 1:4000; Novus Biologicals).

Techniques: Real-time Polymerase Chain Reaction, Comparison, Control, Western Blot, Immunohistochemistry

Journal: The American journal of pathology

Article Title: Choroidal Involution Is Associated with a Progressive Degeneration of the Outer Retinal Function in a Model of Retinopathy of Prematurity: Early Role for IL-1β.

doi: 10.1016/j.ajpath.2016.08.004

Figure Lengend Snippet: Figure 2 IL-1b Q61 has a negative impact on endothelial cells in vitro, ex vivo, and in vivo. A: Graph showing the percentage of survival on rat brain endothelial cells (RBECs) cultured with increased concentrations of rat IL-1b (from 0 to 5000 pmol/L). MTT assay was performed to measure cell viability. IL-1b dose dependently reduced RBEC viability. B: Representative images from choroidal explants cultured in absence or presence of IL-1b (from 0.5 to 5000 pmol/L) and IL-1ra (Kineret, 10 mg/mL) for 48 hours; explants were also stained with fluorescein-conjugated lectin (green) for specific detection of endothelium. Bright-field and fluorescent images were taken. Choroidal sprouting was dose dependently inhibited by IL-1b. The increase in sprouting in the control group was diminished by 50 and 5000 pmol/L IL-1b. IL-1ra coincubation effectively restored choroidal sprouting. C and D: Analyses based on bright-field and fluorescent images revealed equivalent observations. E and F: Representative P14 images of choroid thicknesses from rats intravitreally treated with vehicle (control), IL-1b (1 mL at 5 ng/mL on P7 and P10), or IL-1b plus IL-ra (Kineret, 20 mg/kg twice a day from P7 to P14). Compared to the control group (27.73 2.39 mm), rats that received intravitreous injection of IL-1b showed significantly thinner choroid (14.99 2.47 mm), whereas IL-raetreated animals showed a preservation of choroidal thickness, as seen in the graph (25.64 1.66 mm). Brackets delineate choroidal thickness. Data are expressed as means SEM Q62 (-). n Z 4 to 5 experiments (-). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Scale bar Z 20 mm (E) Q63 . P, postnatal day.

Article Snippet: Because both control and treated eyes were embedded in the same mode and stained with the same procedure, the differences induced by artifacts are minimized. ajp.amjpathol.org - The American Journal of Pathology 496 18 October 2016 5:27 pm EO: AJP16_0032 Q36 Q37 1⁄2T2 Q38 Q39 1⁄2F1 1⁄2F2 Q7 Choroidal Involution and Its Long-Term Impact on Retinal Degeneration 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 Western Blot Protein extracts (40 mg) were loaded, separated by voltage potential, and transferred to polyscreen polyvinylidene difluoride transfer membrane (PerkinElmer), as previously reported.23 Respective proteins were revealed by goat antie IL-1b polyclonal antibody (AF-501-NA, 1:500; R&D Systems) or mouse antieb-actin monoclonal antibody (NB600-501, 1:4000; Novus Biologicals).

Techniques: In Vitro, Ex Vivo, In Vivo, Cell Culture, MTT Assay, Staining, Control, Injection, Preserving

Journal: The American journal of pathology

Article Title: Choroidal Involution Is Associated with a Progressive Degeneration of the Outer Retinal Function in a Model of Retinopathy of Prematurity: Early Role for IL-1β.

doi: 10.1016/j.ajpath.2016.08.004

Figure Lengend Snippet: Figure 9 Schematic diagrams of long-term dysfunctionality of the outer retina from animals subjected to oxygen-induced retinopathy (OIR). A: During early OIR, the choroid starts to become thinner (involution). Unlike inner retinal vascula- ture degeneration, where revascularization occurs, choroidal involution persists throughout life. Accordingly, subretinal hypoxia is detected after choroidal involution. Pleomorphic retinal pigment epithelial (RPE) cells begin to appear on P60 (adolescence), and their number continues to augment when OIR animals age. Meanwhile, a large number of mitochondria lacking cristae (termed ghost mitochondria) appear on P150 (adulthood). Starting on P60 (adolescence), fewer photoreceptors are observed in OIR animals. Photoreceptor population continues to decline as the animal ages. Subsequently, photoreceptor function follows a similar trenddOIR animals elicit marked attenuated photoreceptor responses starting on P60 (adolescence) and continue to deteriorate as they age. B: A schematic represen- tation of choroidal involution associated with OIR and subsequent long-term degeneration of the outer retina. IL-1b is produced by RPE cells in response to retinopathy. Acting in a paracrine manner, IL-1b triggers involution of the choroid (the sole blood supply to RPE and photoreceptors), and subsequently, severe hypoxia in the outer retina. As choroidal involution persists, RPE cells gradually become pleomorphic and ghost mito- chondria (within RPE) start to emerge, likely because of metabolic imbalance. With a global reduction in oxygen and nutrients, as well as dysfunctional RPE cells, photoreceptor population and function also take a downturn. Early antieIL- 1b treatment rescues choroid integrity, thereby preventing outer retinal hypoxia, RPE abnormal- ities, and photoreceptor population and functional decline. P, postnatal day; ROP, retinopathy of prematurity.

Article Snippet: Because both control and treated eyes were embedded in the same mode and stained with the same procedure, the differences induced by artifacts are minimized. ajp.amjpathol.org - The American Journal of Pathology 496 18 October 2016 5:27 pm EO: AJP16_0032 Q36 Q37 1⁄2T2 Q38 Q39 1⁄2F1 1⁄2F2 Q7 Choroidal Involution and Its Long-Term Impact on Retinal Degeneration 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 Western Blot Protein extracts (40 mg) were loaded, separated by voltage potential, and transferred to polyscreen polyvinylidene difluoride transfer membrane (PerkinElmer), as previously reported.23 Respective proteins were revealed by goat antie IL-1b polyclonal antibody (AF-501-NA, 1:500; R&D Systems) or mouse antieb-actin monoclonal antibody (NB600-501, 1:4000; Novus Biologicals).

Techniques: Produced, Functional Assay