Journal: bioRxiv

Article Title: 4-Methylumbelliferone Restores Age-Related Changes in Perineuronal Nets, Memory and Neuroinflammation

doi: 10.64898/2026.01.30.702721

Figure Lengend Snippet: (A) Representative images of CD45 (red), Iba1 (green) and DAPI (blue) staining in the indicated regions. Arrows mark CD45 + Iba1 − cells and the insets show a magnified view of one such cell. (B) Quantification of CD45 + Iba1 − cells per mm 2 in the indicated areas, showing increased number of PICs in the corpus callosum with aging, which was reduced to young adult levels after 4-MU treatment. Data are presented as mean ± SD and were analyzed using ordinary one-way ANOVA; **p < 0.01, **** p < 0.0001.

Article Snippet: Sections were incubated overnight at 4 °C with primary antibodies against GFAP (chicken, 1:800, ab4674, Abcam), Iba1 (goat, 1:800, 019-19741, Fujifilm), CD45 (rabbit, 1:300, bs-10599R, Bioss), and biotinylated WFA.

Techniques: Staining

Journal: eLife

Article Title: Vascular endothelial-specific loss of TGF-beta signaling as a model for choroidal neovascularization and central nervous system vascular inflammation

doi: 10.7554/eLife.107018

Figure Lengend Snippet: ( A ) Cdh5CreER;Tgfbr1 CKO/- retinas showing CNV (white arrows in central panels) and vascular invasion of the outer nuclear layer (white arrows in lower panels), both with associated CD45+ immune cells. ( B ) Cdh5CreER;Tgfbr2 CKO/- retinas showing CNV (white arrows in central panels) and an anastomosis between retinal and choroidal vasculatures (white arrows in lower panels), with intraretinal EC marker CLDN5, choroidal EC marker PLVAP, and pan-vascular marker COL4 (collagen4). ( C ) Upper right panels, in the Cdh5CreER;Tgfbr1 CKO/- retina, CNV (white arrows) is derived from choroidal vasculature, marked by PLVAP. Lower right panels, CNV (white arrows) is present in the subretinal space, i.e., on the retinal side of the RPE, which is marked by RPE65. Abbreviations: Ch., choroid; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer; RPE, retinal pigment epithelium. The ages of the mice are indicated in postnatal days (P) for this and all other figures. Scale bars, 100 µm.

Article Snippet: The following antibodies were used for tissue immunohistochemistry: rat mAb anti-mouse PLVAP/MECA-32 (BD Biosciences 553849); rat mAb anti-mouse CD31 (BD Biosciences 553370); rat anti-mouse ICAM-1 (Invitrogen 14-0542-82); rat mAb anti-mouse F4/80 (Bio-Rad MCA497G); rat mAb anti-mouse CD206 (Bio-Rad MCA2235); rat mAb anti-mouse PU.1/Spi-1 (R&D Systems MAB7124); mouse mAb anti-alpha SMA, Cy3 conjugate (Sigma-Aldrich C6198); mouse mAb anti-CLDN5, Alexa Fluor 488 conjugate (Thermo Fisher Scientific 352588); mouse mAb anti-RPE65, Dylight 550 conjugate (Invitrogen MA5-16044); rabbit polyclonal anti-Collagen IV (Novus Biologicals NB120-6586); rabbit polyclonal anti-NG2 Chondroitin Sulfate Proteoglycan (Millipore AB5320); rabbit mAb anti-ASC/TMS1 (Cell Signaling 67824S); rabbit mAb anti-cleaved Caspase-3 (Cell Signaling 9664S); rabbit mAb anti-HIF-1alpha (Cell Signaling 36169S); rabbit mAb anti-P-SMAD1/5/9 (Cell Signaling 13820S); Armenian hamster mAb anti-CD3e (Invitrogen 14-0031-82); goat polyclonal anti-mouse CD45 (R&D Systems AF114); goat polyclonal anti-Iba1 (Novus Biologicals NB100-1028); chicken polyclonal anti-GFP (Aves Labs GFP-1020); rabbit mAb anti-NFkappaB NF-κB p65 (D14E12; Cell Signaling Technology 8242S); rabbit mAb anti-Integrin alpha 2 (ITGA2; clone GEB, BosterBio M01933); rabbit mAb anti-Integrin alpha 4 (ITGA4; D2E1; Cell Signaling Technology 8440); rabbit mAb anti-TOX/TOX2 (E6G5O; Cell Signaling Technology 36778S).

Techniques: Marker, Derivative Assay

Journal: eLife

Article Title: Vascular endothelial-specific loss of TGF-beta signaling as a model for choroidal neovascularization and central nervous system vascular inflammation

doi: 10.7554/eLife.107018

Figure Lengend Snippet: ( A ) Phenotypically wild-type (WT) control retina. ( B ) Cdh5CreER;Tgfbr1 CKO/- retina. Eyes from mice at ~P90 were fresh frozen, sectioned, and immunostained. RPE65 marks the retinal pigment epithelium (RPE). In the Cdh5CreER;Tgfbr1 CKO/- retina in ( B ), PECAM1+ ECs and associated RPE cells can be seen protruding into the subretinal space in an ~200 µm wide region (white arrows). ( C ) Quantification of CNV tufts in frozen sections of adult Cdh5CreER;Tgfbr1 CKO/- retinas, Cdh5CreER;Tgfbr2 CKO/- retinas, Ndp KO retinas, and age-matched control retinas. Each data point represents a single whole eye section. CNV tufts were seen only in Cdh5CreER;Tgfbr1 CKO/- retinas. ( D ) Retina flatmounts from Cdh5CreER;Tgfbr1 CKO/- ~P90 mice show scattered CD45+ cells (upper panel) and variably sized zones of CNV with vessel-associated CD45+ cells (lower panel). The stacked Z-planes of the images are at the level of photoreceptors and RPE. Retinas were mounted photoreceptor side up. All other retina flatmount images were mounted ganglion cell side up. The confocal microscope collects images from above. Abbreviations: Ch., choroid; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer; P, postnatal day. Scale bars, 100 µm.

Article Snippet: The following antibodies were used for tissue immunohistochemistry: rat mAb anti-mouse PLVAP/MECA-32 (BD Biosciences 553849); rat mAb anti-mouse CD31 (BD Biosciences 553370); rat anti-mouse ICAM-1 (Invitrogen 14-0542-82); rat mAb anti-mouse F4/80 (Bio-Rad MCA497G); rat mAb anti-mouse CD206 (Bio-Rad MCA2235); rat mAb anti-mouse PU.1/Spi-1 (R&D Systems MAB7124); mouse mAb anti-alpha SMA, Cy3 conjugate (Sigma-Aldrich C6198); mouse mAb anti-CLDN5, Alexa Fluor 488 conjugate (Thermo Fisher Scientific 352588); mouse mAb anti-RPE65, Dylight 550 conjugate (Invitrogen MA5-16044); rabbit polyclonal anti-Collagen IV (Novus Biologicals NB120-6586); rabbit polyclonal anti-NG2 Chondroitin Sulfate Proteoglycan (Millipore AB5320); rabbit mAb anti-ASC/TMS1 (Cell Signaling 67824S); rabbit mAb anti-cleaved Caspase-3 (Cell Signaling 9664S); rabbit mAb anti-HIF-1alpha (Cell Signaling 36169S); rabbit mAb anti-P-SMAD1/5/9 (Cell Signaling 13820S); Armenian hamster mAb anti-CD3e (Invitrogen 14-0031-82); goat polyclonal anti-mouse CD45 (R&D Systems AF114); goat polyclonal anti-Iba1 (Novus Biologicals NB100-1028); chicken polyclonal anti-GFP (Aves Labs GFP-1020); rabbit mAb anti-NFkappaB NF-κB p65 (D14E12; Cell Signaling Technology 8242S); rabbit mAb anti-Integrin alpha 2 (ITGA2; clone GEB, BosterBio M01933); rabbit mAb anti-Integrin alpha 4 (ITGA4; D2E1; Cell Signaling Technology 8440); rabbit mAb anti-TOX/TOX2 (E6G5O; Cell Signaling Technology 36778S).

Techniques: Control, Microscopy

Journal: eLife

Article Title: Vascular endothelial-specific loss of TGF-beta signaling as a model for choroidal neovascularization and central nervous system vascular inflammation

doi: 10.7554/eLife.107018

Figure Lengend Snippet: Collagen-4 (COL4) marks ECM, including perivascular ECM. ICAM1 is unchanged. P14 corresponds to the peak of CD45+ cell counts in the Cdh5CreER;Tgfbr1 CKO/- retina. Scale bar, 50 µm.

Article Snippet: The following antibodies were used for tissue immunohistochemistry: rat mAb anti-mouse PLVAP/MECA-32 (BD Biosciences 553849); rat mAb anti-mouse CD31 (BD Biosciences 553370); rat anti-mouse ICAM-1 (Invitrogen 14-0542-82); rat mAb anti-mouse F4/80 (Bio-Rad MCA497G); rat mAb anti-mouse CD206 (Bio-Rad MCA2235); rat mAb anti-mouse PU.1/Spi-1 (R&D Systems MAB7124); mouse mAb anti-alpha SMA, Cy3 conjugate (Sigma-Aldrich C6198); mouse mAb anti-CLDN5, Alexa Fluor 488 conjugate (Thermo Fisher Scientific 352588); mouse mAb anti-RPE65, Dylight 550 conjugate (Invitrogen MA5-16044); rabbit polyclonal anti-Collagen IV (Novus Biologicals NB120-6586); rabbit polyclonal anti-NG2 Chondroitin Sulfate Proteoglycan (Millipore AB5320); rabbit mAb anti-ASC/TMS1 (Cell Signaling 67824S); rabbit mAb anti-cleaved Caspase-3 (Cell Signaling 9664S); rabbit mAb anti-HIF-1alpha (Cell Signaling 36169S); rabbit mAb anti-P-SMAD1/5/9 (Cell Signaling 13820S); Armenian hamster mAb anti-CD3e (Invitrogen 14-0031-82); goat polyclonal anti-mouse CD45 (R&D Systems AF114); goat polyclonal anti-Iba1 (Novus Biologicals NB100-1028); chicken polyclonal anti-GFP (Aves Labs GFP-1020); rabbit mAb anti-NFkappaB NF-κB p65 (D14E12; Cell Signaling Technology 8242S); rabbit mAb anti-Integrin alpha 2 (ITGA2; clone GEB, BosterBio M01933); rabbit mAb anti-Integrin alpha 4 (ITGA4; D2E1; Cell Signaling Technology 8440); rabbit mAb anti-TOX/TOX2 (E6G5O; Cell Signaling Technology 36778S).

Techniques:

Journal: eLife

Article Title: Vascular endothelial-specific loss of TGF-beta signaling as a model for choroidal neovascularization and central nervous system vascular inflammation

doi: 10.7554/eLife.107018

Figure Lengend Snippet: ( A, B ) Co-localization of the nuclear-localized GFP reporter with the EC transcription factor ERG and non-co-localization with the macrophage marker CD45 in ( A ) a flatmount of choroid and in ( B ) a vibratome section of small intestine. In the rightmost panel, GFP and CD45 are seen to have distinct localizations in both tissues. ( C ) Retina flatmounts showing non-overlapping patterns of GFP (elongated EC nuclei) and CD45 (microglia). Scale bars, 100 µm.

Article Snippet: The following antibodies were used for tissue immunohistochemistry: rat mAb anti-mouse PLVAP/MECA-32 (BD Biosciences 553849); rat mAb anti-mouse CD31 (BD Biosciences 553370); rat anti-mouse ICAM-1 (Invitrogen 14-0542-82); rat mAb anti-mouse F4/80 (Bio-Rad MCA497G); rat mAb anti-mouse CD206 (Bio-Rad MCA2235); rat mAb anti-mouse PU.1/Spi-1 (R&D Systems MAB7124); mouse mAb anti-alpha SMA, Cy3 conjugate (Sigma-Aldrich C6198); mouse mAb anti-CLDN5, Alexa Fluor 488 conjugate (Thermo Fisher Scientific 352588); mouse mAb anti-RPE65, Dylight 550 conjugate (Invitrogen MA5-16044); rabbit polyclonal anti-Collagen IV (Novus Biologicals NB120-6586); rabbit polyclonal anti-NG2 Chondroitin Sulfate Proteoglycan (Millipore AB5320); rabbit mAb anti-ASC/TMS1 (Cell Signaling 67824S); rabbit mAb anti-cleaved Caspase-3 (Cell Signaling 9664S); rabbit mAb anti-HIF-1alpha (Cell Signaling 36169S); rabbit mAb anti-P-SMAD1/5/9 (Cell Signaling 13820S); Armenian hamster mAb anti-CD3e (Invitrogen 14-0031-82); goat polyclonal anti-mouse CD45 (R&D Systems AF114); goat polyclonal anti-Iba1 (Novus Biologicals NB100-1028); chicken polyclonal anti-GFP (Aves Labs GFP-1020); rabbit mAb anti-NFkappaB NF-κB p65 (D14E12; Cell Signaling Technology 8242S); rabbit mAb anti-Integrin alpha 2 (ITGA2; clone GEB, BosterBio M01933); rabbit mAb anti-Integrin alpha 4 (ITGA4; D2E1; Cell Signaling Technology 8440); rabbit mAb anti-TOX/TOX2 (E6G5O; Cell Signaling Technology 36778S).

Techniques: Marker

Journal: eLife

Article Title: Vascular endothelial-specific loss of TGF-beta signaling as a model for choroidal neovascularization and central nervous system vascular inflammation

doi: 10.7554/eLife.107018

Figure Lengend Snippet: A retina flatmount from a P17 Cdh5CreER;Tgfbr1 CKO/- ;Rosa26-LSL-SUN1-sfGFP-6xmyc mouse shows the phenotype of CD45+ immune cell accumulation following loss of TGF-beta signaling. GFP expressed from the LSL reporter co-localizes with ERG and shows no co-localization with CD45. Scale bar, 100 µm.

Article Snippet: The following antibodies were used for tissue immunohistochemistry: rat mAb anti-mouse PLVAP/MECA-32 (BD Biosciences 553849); rat mAb anti-mouse CD31 (BD Biosciences 553370); rat anti-mouse ICAM-1 (Invitrogen 14-0542-82); rat mAb anti-mouse F4/80 (Bio-Rad MCA497G); rat mAb anti-mouse CD206 (Bio-Rad MCA2235); rat mAb anti-mouse PU.1/Spi-1 (R&D Systems MAB7124); mouse mAb anti-alpha SMA, Cy3 conjugate (Sigma-Aldrich C6198); mouse mAb anti-CLDN5, Alexa Fluor 488 conjugate (Thermo Fisher Scientific 352588); mouse mAb anti-RPE65, Dylight 550 conjugate (Invitrogen MA5-16044); rabbit polyclonal anti-Collagen IV (Novus Biologicals NB120-6586); rabbit polyclonal anti-NG2 Chondroitin Sulfate Proteoglycan (Millipore AB5320); rabbit mAb anti-ASC/TMS1 (Cell Signaling 67824S); rabbit mAb anti-cleaved Caspase-3 (Cell Signaling 9664S); rabbit mAb anti-HIF-1alpha (Cell Signaling 36169S); rabbit mAb anti-P-SMAD1/5/9 (Cell Signaling 13820S); Armenian hamster mAb anti-CD3e (Invitrogen 14-0031-82); goat polyclonal anti-mouse CD45 (R&D Systems AF114); goat polyclonal anti-Iba1 (Novus Biologicals NB100-1028); chicken polyclonal anti-GFP (Aves Labs GFP-1020); rabbit mAb anti-NFkappaB NF-κB p65 (D14E12; Cell Signaling Technology 8242S); rabbit mAb anti-Integrin alpha 2 (ITGA2; clone GEB, BosterBio M01933); rabbit mAb anti-Integrin alpha 4 (ITGA4; D2E1; Cell Signaling Technology 8440); rabbit mAb anti-TOX/TOX2 (E6G5O; Cell Signaling Technology 36778S).

Techniques:

Journal: eLife

Article Title: Vascular endothelial-specific loss of TGF-beta signaling as a model for choroidal neovascularization and central nervous system vascular inflammation

doi: 10.7554/eLife.107018

Figure Lengend Snippet: ( A ) Microglia and retinal endothelial cells (ECs) were visualized by immunostaining for CD45 and PECAM1 (upper two panels). By visual inspection, the distributions of the two cell types appear to be uncorrelated. For quantification, microglia were visualized by immunostaining for CD45 (plasma membrane) and ASC1 (nucleus) (lower two panels). The regions in the white squares in the left two panels are shown enlarged in the right two panels. ( B ) Quantification of microglial density in each of the three layers of the inner retina, the regions where microglia are found. Bars represent mean ± standard deviation. RGC, retinal ganglion cell layer; IPL, inner plexiform layer; OPL, outer plexiform layer. Scale bars in ( A ): left, 100 µm, right, 50 µm.

Article Snippet: The following antibodies were used for tissue immunohistochemistry: rat mAb anti-mouse PLVAP/MECA-32 (BD Biosciences 553849); rat mAb anti-mouse CD31 (BD Biosciences 553370); rat anti-mouse ICAM-1 (Invitrogen 14-0542-82); rat mAb anti-mouse F4/80 (Bio-Rad MCA497G); rat mAb anti-mouse CD206 (Bio-Rad MCA2235); rat mAb anti-mouse PU.1/Spi-1 (R&D Systems MAB7124); mouse mAb anti-alpha SMA, Cy3 conjugate (Sigma-Aldrich C6198); mouse mAb anti-CLDN5, Alexa Fluor 488 conjugate (Thermo Fisher Scientific 352588); mouse mAb anti-RPE65, Dylight 550 conjugate (Invitrogen MA5-16044); rabbit polyclonal anti-Collagen IV (Novus Biologicals NB120-6586); rabbit polyclonal anti-NG2 Chondroitin Sulfate Proteoglycan (Millipore AB5320); rabbit mAb anti-ASC/TMS1 (Cell Signaling 67824S); rabbit mAb anti-cleaved Caspase-3 (Cell Signaling 9664S); rabbit mAb anti-HIF-1alpha (Cell Signaling 36169S); rabbit mAb anti-P-SMAD1/5/9 (Cell Signaling 13820S); Armenian hamster mAb anti-CD3e (Invitrogen 14-0031-82); goat polyclonal anti-mouse CD45 (R&D Systems AF114); goat polyclonal anti-Iba1 (Novus Biologicals NB100-1028); chicken polyclonal anti-GFP (Aves Labs GFP-1020); rabbit mAb anti-NFkappaB NF-κB p65 (D14E12; Cell Signaling Technology 8242S); rabbit mAb anti-Integrin alpha 2 (ITGA2; clone GEB, BosterBio M01933); rabbit mAb anti-Integrin alpha 4 (ITGA4; D2E1; Cell Signaling Technology 8440); rabbit mAb anti-TOX/TOX2 (E6G5O; Cell Signaling Technology 36778S).

Techniques: Immunostaining, Clinical Proteomics, Membrane, Standard Deviation

Journal: eLife

Article Title: Vascular endothelial-specific loss of TGF-beta signaling as a model for choroidal neovascularization and central nervous system vascular inflammation

doi: 10.7554/eLife.107018

Figure Lengend Snippet: ( A ) After removing the retina, choroid flatmounts (sclera, choroidal vasculature, and retinal pigment epithelium [RPE]) were imaged from the RPE side. ( B ) The number of CD45+ cells in choroid flatmounts. Bars represent mean ± standard deviation, and p-values, calculated using the Wilcoxon rank-sum test, are shown as *<0.05, **<0.01, ***<0.001, and ****<0.0001. Scale bar, 100 µm.

Article Snippet: The following antibodies were used for tissue immunohistochemistry: rat mAb anti-mouse PLVAP/MECA-32 (BD Biosciences 553849); rat mAb anti-mouse CD31 (BD Biosciences 553370); rat anti-mouse ICAM-1 (Invitrogen 14-0542-82); rat mAb anti-mouse F4/80 (Bio-Rad MCA497G); rat mAb anti-mouse CD206 (Bio-Rad MCA2235); rat mAb anti-mouse PU.1/Spi-1 (R&D Systems MAB7124); mouse mAb anti-alpha SMA, Cy3 conjugate (Sigma-Aldrich C6198); mouse mAb anti-CLDN5, Alexa Fluor 488 conjugate (Thermo Fisher Scientific 352588); mouse mAb anti-RPE65, Dylight 550 conjugate (Invitrogen MA5-16044); rabbit polyclonal anti-Collagen IV (Novus Biologicals NB120-6586); rabbit polyclonal anti-NG2 Chondroitin Sulfate Proteoglycan (Millipore AB5320); rabbit mAb anti-ASC/TMS1 (Cell Signaling 67824S); rabbit mAb anti-cleaved Caspase-3 (Cell Signaling 9664S); rabbit mAb anti-HIF-1alpha (Cell Signaling 36169S); rabbit mAb anti-P-SMAD1/5/9 (Cell Signaling 13820S); Armenian hamster mAb anti-CD3e (Invitrogen 14-0031-82); goat polyclonal anti-mouse CD45 (R&D Systems AF114); goat polyclonal anti-Iba1 (Novus Biologicals NB100-1028); chicken polyclonal anti-GFP (Aves Labs GFP-1020); rabbit mAb anti-NFkappaB NF-κB p65 (D14E12; Cell Signaling Technology 8242S); rabbit mAb anti-Integrin alpha 2 (ITGA2; clone GEB, BosterBio M01933); rabbit mAb anti-Integrin alpha 4 (ITGA4; D2E1; Cell Signaling Technology 8440); rabbit mAb anti-TOX/TOX2 (E6G5O; Cell Signaling Technology 36778S).

Techniques: Standard Deviation

Journal: eLife

Article Title: Vascular endothelial-specific loss of TGF-beta signaling as a model for choroidal neovascularization and central nervous system vascular inflammation

doi: 10.7554/eLife.107018

Figure Lengend Snippet: ( A ) Retina flatmounts from mice with the indicated genotypes were immunostained for cleaved Caspase 3, CD45, and PECAM1. In Cdh5CreER;Tgfbr1 CKO/- and Cdh5CreER;Tgfbr1 CKO/- ;Tgfbr2 CKO/- retinas, more than 20% of CD45+ cells are also positive for cleaved caspase 3. ( B ) Quantifying the number of cleaved caspase 3+ cells in control, Fzd4 -/- , and Cdh5CreER;Tgfbr1 CKO/- retinas. Bars represent mean ± standard deviation, and p-values, calculated using the Wilcoxon rank-sum test, are shown as *<0.05, **<0.01, ***<0.001, and ****<0.0001. n.s., not significant. Scale bar, 100 µm.

Article Snippet: The following antibodies were used for tissue immunohistochemistry: rat mAb anti-mouse PLVAP/MECA-32 (BD Biosciences 553849); rat mAb anti-mouse CD31 (BD Biosciences 553370); rat anti-mouse ICAM-1 (Invitrogen 14-0542-82); rat mAb anti-mouse F4/80 (Bio-Rad MCA497G); rat mAb anti-mouse CD206 (Bio-Rad MCA2235); rat mAb anti-mouse PU.1/Spi-1 (R&D Systems MAB7124); mouse mAb anti-alpha SMA, Cy3 conjugate (Sigma-Aldrich C6198); mouse mAb anti-CLDN5, Alexa Fluor 488 conjugate (Thermo Fisher Scientific 352588); mouse mAb anti-RPE65, Dylight 550 conjugate (Invitrogen MA5-16044); rabbit polyclonal anti-Collagen IV (Novus Biologicals NB120-6586); rabbit polyclonal anti-NG2 Chondroitin Sulfate Proteoglycan (Millipore AB5320); rabbit mAb anti-ASC/TMS1 (Cell Signaling 67824S); rabbit mAb anti-cleaved Caspase-3 (Cell Signaling 9664S); rabbit mAb anti-HIF-1alpha (Cell Signaling 36169S); rabbit mAb anti-P-SMAD1/5/9 (Cell Signaling 13820S); Armenian hamster mAb anti-CD3e (Invitrogen 14-0031-82); goat polyclonal anti-mouse CD45 (R&D Systems AF114); goat polyclonal anti-Iba1 (Novus Biologicals NB100-1028); chicken polyclonal anti-GFP (Aves Labs GFP-1020); rabbit mAb anti-NFkappaB NF-κB p65 (D14E12; Cell Signaling Technology 8242S); rabbit mAb anti-Integrin alpha 2 (ITGA2; clone GEB, BosterBio M01933); rabbit mAb anti-Integrin alpha 4 (ITGA4; D2E1; Cell Signaling Technology 8440); rabbit mAb anti-TOX/TOX2 (E6G5O; Cell Signaling Technology 36778S).

Techniques: Control, Standard Deviation

Journal: eLife

Article Title: Vascular endothelial-specific loss of TGF-beta signaling as a model for choroidal neovascularization and central nervous system vascular inflammation

doi: 10.7554/eLife.107018

Figure Lengend Snippet: ( A ) Sections of control and Vsx2-Cre;Vegfa CKO/CKO retinas immunostained with anti-PECAM1 to visualize the vasculature. ( B ) False color images of control and Vsx2-Cre;Vegfa CKO/CKO flatmount retinas showing a stacked Z-series color-coded by the depth of PECAM1 immunostained vasculature. Blue, vitreal surface; green, inner plexiform layer; red, outer plexiform layer. ( C ) Control and Vsx2-Cre;Vegfa CKO/CKO flatmount retinas immunostained with anti-PECAM1. ( D ) Control and Vsx2-Cre;Vegfa CKO/CKO flatmount retinas immunostained for ASC and CD45. ( E ) Quantification of CD45+/ASC+ cells. Scale bars, ( A ), ( B ), and ( D ), 100 µm; ( C ) 500 µm.

Article Snippet: The following antibodies were used for tissue immunohistochemistry: rat mAb anti-mouse PLVAP/MECA-32 (BD Biosciences 553849); rat mAb anti-mouse CD31 (BD Biosciences 553370); rat anti-mouse ICAM-1 (Invitrogen 14-0542-82); rat mAb anti-mouse F4/80 (Bio-Rad MCA497G); rat mAb anti-mouse CD206 (Bio-Rad MCA2235); rat mAb anti-mouse PU.1/Spi-1 (R&D Systems MAB7124); mouse mAb anti-alpha SMA, Cy3 conjugate (Sigma-Aldrich C6198); mouse mAb anti-CLDN5, Alexa Fluor 488 conjugate (Thermo Fisher Scientific 352588); mouse mAb anti-RPE65, Dylight 550 conjugate (Invitrogen MA5-16044); rabbit polyclonal anti-Collagen IV (Novus Biologicals NB120-6586); rabbit polyclonal anti-NG2 Chondroitin Sulfate Proteoglycan (Millipore AB5320); rabbit mAb anti-ASC/TMS1 (Cell Signaling 67824S); rabbit mAb anti-cleaved Caspase-3 (Cell Signaling 9664S); rabbit mAb anti-HIF-1alpha (Cell Signaling 36169S); rabbit mAb anti-P-SMAD1/5/9 (Cell Signaling 13820S); Armenian hamster mAb anti-CD3e (Invitrogen 14-0031-82); goat polyclonal anti-mouse CD45 (R&D Systems AF114); goat polyclonal anti-Iba1 (Novus Biologicals NB100-1028); chicken polyclonal anti-GFP (Aves Labs GFP-1020); rabbit mAb anti-NFkappaB NF-κB p65 (D14E12; Cell Signaling Technology 8242S); rabbit mAb anti-Integrin alpha 2 (ITGA2; clone GEB, BosterBio M01933); rabbit mAb anti-Integrin alpha 4 (ITGA4; D2E1; Cell Signaling Technology 8440); rabbit mAb anti-TOX/TOX2 (E6G5O; Cell Signaling Technology 36778S).

Techniques: Control

Journal: eLife

Article Title: Vascular endothelial-specific loss of TGF-beta signaling as a model for choroidal neovascularization and central nervous system vascular inflammation

doi: 10.7554/eLife.107018

Figure Lengend Snippet: ( A ) Upper six panels show a control retina flatmount. Lower six panels show a Cdh5CreER;Tgfbr1 CKO/+ ;Tgfbr2 CKO/- retina flatmount. ASC and CD45 label immune cells, including microglia. The Z-plane is indicated by the numbers at the bottom of each image. The nerve fiber layer (Z-plane 3) and the inner plexiform layer (Z-planes 10–11) are shown schematically in ( B ) and ( C ). ( B and C ) Retinal schematics showing the relationship of the vasculature and the three retinal layers. Confocal Z-planes are numbered at right. ( D ) Immune cells and venous ECs in control and Cdh5CreER;Tgfbr1 CKO/- retinas. In the lower image, three of the ‘impressions’ of CD45+ immune in the distribution of PECAM1 on the EC surface are highlighted with white arrows. A, artery; V, vein. NFL, nerve fiber layer; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; IS/OS, inner segment/outer segment. Scale bars in ( A ), 100 µm. Scale bar in ( D ), 50 µm.

Article Snippet: The following antibodies were used for tissue immunohistochemistry: rat mAb anti-mouse PLVAP/MECA-32 (BD Biosciences 553849); rat mAb anti-mouse CD31 (BD Biosciences 553370); rat anti-mouse ICAM-1 (Invitrogen 14-0542-82); rat mAb anti-mouse F4/80 (Bio-Rad MCA497G); rat mAb anti-mouse CD206 (Bio-Rad MCA2235); rat mAb anti-mouse PU.1/Spi-1 (R&D Systems MAB7124); mouse mAb anti-alpha SMA, Cy3 conjugate (Sigma-Aldrich C6198); mouse mAb anti-CLDN5, Alexa Fluor 488 conjugate (Thermo Fisher Scientific 352588); mouse mAb anti-RPE65, Dylight 550 conjugate (Invitrogen MA5-16044); rabbit polyclonal anti-Collagen IV (Novus Biologicals NB120-6586); rabbit polyclonal anti-NG2 Chondroitin Sulfate Proteoglycan (Millipore AB5320); rabbit mAb anti-ASC/TMS1 (Cell Signaling 67824S); rabbit mAb anti-cleaved Caspase-3 (Cell Signaling 9664S); rabbit mAb anti-HIF-1alpha (Cell Signaling 36169S); rabbit mAb anti-P-SMAD1/5/9 (Cell Signaling 13820S); Armenian hamster mAb anti-CD3e (Invitrogen 14-0031-82); goat polyclonal anti-mouse CD45 (R&D Systems AF114); goat polyclonal anti-Iba1 (Novus Biologicals NB100-1028); chicken polyclonal anti-GFP (Aves Labs GFP-1020); rabbit mAb anti-NFkappaB NF-κB p65 (D14E12; Cell Signaling Technology 8242S); rabbit mAb anti-Integrin alpha 2 (ITGA2; clone GEB, BosterBio M01933); rabbit mAb anti-Integrin alpha 4 (ITGA4; D2E1; Cell Signaling Technology 8440); rabbit mAb anti-TOX/TOX2 (E6G5O; Cell Signaling Technology 36778S).

Techniques: Control

Journal: eLife

Article Title: Vascular endothelial-specific loss of TGF-beta signaling as a model for choroidal neovascularization and central nervous system vascular inflammation

doi: 10.7554/eLife.107018

Figure Lengend Snippet: ( A ) Upper three panels show a control retina flatmount with sparse CD45+ microglia and PECAM1+ vasculature, imaged at three Z-planes (indicated by the numbers in the lower left corner of each image) corresponding to the NFL, IPL, and OPL. The lower nine panels show a series of Z-planes through the full thickness of a Cdh5CreER;Tgfbr1 CKO/- retina flatmount with many CD45+ immune cells in contact with the vasculature. ( B and C ) Retinal schematics showing the relationship of the vasculature and the three retinal layers. Confocal Z-planes are numbered at right. NFL, nerve fiber layer; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; IS/OS, inner segment/outer segment. Scale bars in ( A ), 100 µm.

Article Snippet: The following antibodies were used for tissue immunohistochemistry: rat mAb anti-mouse PLVAP/MECA-32 (BD Biosciences 553849); rat mAb anti-mouse CD31 (BD Biosciences 553370); rat anti-mouse ICAM-1 (Invitrogen 14-0542-82); rat mAb anti-mouse F4/80 (Bio-Rad MCA497G); rat mAb anti-mouse CD206 (Bio-Rad MCA2235); rat mAb anti-mouse PU.1/Spi-1 (R&D Systems MAB7124); mouse mAb anti-alpha SMA, Cy3 conjugate (Sigma-Aldrich C6198); mouse mAb anti-CLDN5, Alexa Fluor 488 conjugate (Thermo Fisher Scientific 352588); mouse mAb anti-RPE65, Dylight 550 conjugate (Invitrogen MA5-16044); rabbit polyclonal anti-Collagen IV (Novus Biologicals NB120-6586); rabbit polyclonal anti-NG2 Chondroitin Sulfate Proteoglycan (Millipore AB5320); rabbit mAb anti-ASC/TMS1 (Cell Signaling 67824S); rabbit mAb anti-cleaved Caspase-3 (Cell Signaling 9664S); rabbit mAb anti-HIF-1alpha (Cell Signaling 36169S); rabbit mAb anti-P-SMAD1/5/9 (Cell Signaling 13820S); Armenian hamster mAb anti-CD3e (Invitrogen 14-0031-82); goat polyclonal anti-mouse CD45 (R&D Systems AF114); goat polyclonal anti-Iba1 (Novus Biologicals NB100-1028); chicken polyclonal anti-GFP (Aves Labs GFP-1020); rabbit mAb anti-NFkappaB NF-κB p65 (D14E12; Cell Signaling Technology 8242S); rabbit mAb anti-Integrin alpha 2 (ITGA2; clone GEB, BosterBio M01933); rabbit mAb anti-Integrin alpha 4 (ITGA4; D2E1; Cell Signaling Technology 8440); rabbit mAb anti-TOX/TOX2 (E6G5O; Cell Signaling Technology 36778S).

Techniques: Control

Journal: eLife

Article Title: Vascular endothelial-specific loss of TGF-beta signaling as a model for choroidal neovascularization and central nervous system vascular inflammation

doi: 10.7554/eLife.107018

Figure Lengend Snippet: ( A ) Endogenous IgG in control and Cdh5CreER;Tgfbr1 CKO/- brains at postnatal day (P)14 and P24. IgG accumulation is minimal in control brains at P14 and P24 but is readily detectable in Cdh5CreER;Tgfbr1 CKO/- brains at P14 but not at P24. ( B ) ECs (visualized with PECAM1) and pericytes (visualized with NG2) in control and Cdh5CreER;Tgfbr1 CKO/- brains at P14. SMA immunostaining visualizes arterioles (continuous staining) and veins (patchy staining) in control and Cdh5CreER;Tgfbr1 CKO/- brains, and a subset of capillary-associated pericytes in Cdh5CreER;Tgfbr1 CKO/- brains. ( C ) Immune cells (CD45+ and F4-80+) in control and Cdh5CreER;Tgfbr1 CKO/- brains at P35. Control brains have minimal numbers of immune cells other than resident microglia. Cdh5CreER;Tgfbr1 CKO/- brains show localized regions with concentrated accumulations of immune cells. White squares marked with letters in the sagittal brain images (upper) are enlarged below. Scale bars, 1 mm for whole brain images and 200 µm for enlarged images.

Article Snippet: The following antibodies were used for tissue immunohistochemistry: rat mAb anti-mouse PLVAP/MECA-32 (BD Biosciences 553849); rat mAb anti-mouse CD31 (BD Biosciences 553370); rat anti-mouse ICAM-1 (Invitrogen 14-0542-82); rat mAb anti-mouse F4/80 (Bio-Rad MCA497G); rat mAb anti-mouse CD206 (Bio-Rad MCA2235); rat mAb anti-mouse PU.1/Spi-1 (R&D Systems MAB7124); mouse mAb anti-alpha SMA, Cy3 conjugate (Sigma-Aldrich C6198); mouse mAb anti-CLDN5, Alexa Fluor 488 conjugate (Thermo Fisher Scientific 352588); mouse mAb anti-RPE65, Dylight 550 conjugate (Invitrogen MA5-16044); rabbit polyclonal anti-Collagen IV (Novus Biologicals NB120-6586); rabbit polyclonal anti-NG2 Chondroitin Sulfate Proteoglycan (Millipore AB5320); rabbit mAb anti-ASC/TMS1 (Cell Signaling 67824S); rabbit mAb anti-cleaved Caspase-3 (Cell Signaling 9664S); rabbit mAb anti-HIF-1alpha (Cell Signaling 36169S); rabbit mAb anti-P-SMAD1/5/9 (Cell Signaling 13820S); Armenian hamster mAb anti-CD3e (Invitrogen 14-0031-82); goat polyclonal anti-mouse CD45 (R&D Systems AF114); goat polyclonal anti-Iba1 (Novus Biologicals NB100-1028); chicken polyclonal anti-GFP (Aves Labs GFP-1020); rabbit mAb anti-NFkappaB NF-κB p65 (D14E12; Cell Signaling Technology 8242S); rabbit mAb anti-Integrin alpha 2 (ITGA2; clone GEB, BosterBio M01933); rabbit mAb anti-Integrin alpha 4 (ITGA4; D2E1; Cell Signaling Technology 8440); rabbit mAb anti-TOX/TOX2 (E6G5O; Cell Signaling Technology 36778S).

Techniques: Control, Immunostaining, Staining