352588

antibody from Invitrogen Antibodies

Targeting: CLDN5 AWAL, BEC1, CPETRL1, TMVCF

Provider product page for 352588

Western blot

ELISA

Immunocytochemistry

Immunoprecipitation

Immunohistochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [53]
Comments [0]
Validations
Immunocytochemistry [1]
Other assay [33]

Submit

Product number: 352588 - Provider product page
Provider: Invitrogen Antibodies
Product name: Claudin 5 Monoclonal Antibody (4C3C2), Alexa Fluor 488
Antibody type: Monoclonal
Antigen: Synthetic peptide
Reactivity: Human, Mouse, Rat
Host: Mouse
Conjugate: Green dye
Isotype: IgG
Antibody clone number: 4C3C2
Vial size: 100 µg
Concentration: 0.5 mg/mL
Storage: 4° C

CLDN5 protein structure - 352588 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL
Conjugate: Green dye

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL
Conjugate: Green dye

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL
Conjugate: Green dye

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL
Conjugate: Green dye

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL
Conjugate: Green dye

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL
Conjugate: Green dye

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL
Conjugate: Green dye

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL
Conjugate: Green dye

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL
Conjugate: Green dye

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL
Conjugate: Green dye

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL
Conjugate: Green dye

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL
Conjugate: Green dye

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL
Conjugate: Green dye

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL
Conjugate: Green dye

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL
Conjugate: Green dye

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Extended Figure 5 Initial lymphatic features of meningeal lymphatic vessels a. Representative images of CCL21 and Lyve-1 labeling of the meningeal lymphatic vessels (scale bar = 10 mum). b-c . Representative images of VE-Cadherin and Lyve-1 staining on meningeal blood vessels (b) and meningeal lymphatic vessels (c), arrowheads point to the VE-Cadherin aggregates; scale bar = 10 mum). d-f . Representative images of Claudin-5 and Lyve-1 staining on meningeal blood (d) and lymphatic (e) vessels, and diaphragm lymphatic vessels (f); arrowheads point to Claudin-5 aggregates (scale bar = 10 mum). g-h. Representative images of integrin-alpha9 and Lyve-1 labeling on skin (g; ear) and meninges whole mount (h). Scale bar = 40 mum. No integrin-alpha9 expressing valves were detected in the meningeal lymphatic vessels. i . Representative low power micrographs (transmission EM) of the meningeal lymphatic vessels (scale bar = 2 mum); (L = lumen; SC = supporting cell; LEC = lymphatic endothelial cell; BEC = sinusal endothelial cell). Red arrowheads point to anchoring filaments. j . Table summarizing morphological features of the lymphatic network in different regions of the meninges and the diaphragm. Diameters are expressed in mum and branching as number of branches per mm of vessel; (mean +- SEM; n = 4 animals each group, *p
Conjugate: Green dye

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig 5 Excess tip cell production in the Sox7, Sox17, and Sox18 EC-specific triple KO ('6-allele loss') at P6. (A-J) Retina flat mounts from control Sox18 +/- (A, C, E, G, I) and 6-allele loss Sox7 CKO/- ;Sox17 CKO/CKO ;Sox18 -/- ;Pdgfb-CreER (B, D, F, H, J) mice at P6. In (I) and (J), retinas were labeled with Alexa488 GS-lectin (green) via intracardiac injection and Alexa595 GS-lectin (red) after postfixation and permeabilization. The dashed lines delineate the boundary between hypoxia/normoxia in (A and B), and perfused/non-perfused vessels in (J). In control retinas, most tip cells (arrows) express higher levels of Dll4 (C) and VEGFR3 (E) than adjacent capillaries. The larger zone of hypoxia in (B) compared to (A) reflects the retarded growth of the vascular plexus. 50-100 mug 4HT was given at P0 and P2. A, artery; V, vein. Scale bar, 200 mum.
Conjugate: Green dye

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 7 HES5 and JAG1 are implicated in endothelial lineage generation from PR-SMCs (II). ( a ) Real-time PCR revealed that after stimulating the cells with immobilized recombinant JAG1 from day 3 during differentiation, eNOS, vWF and Claudin-5 could be further upregulated (*p < 0.05 by Student's t test, n = 3). ( b ) Knockdown JAG1 expression led to the downregulation of eNOS and Claudin-5 (*p < 0.05 by Student's t test, n = 3). ( c ) Western blot analysis confirmed the downregulation of endothelial markers in concert with the JAG1 suppression. Control group refers to the SMC transfected with empty lentiviral vector that underwent the same reprogramming and differentiation protocol. Bands were cropped for the clarity. ( d , e ) Luciferase assay showed the activation of eNOS and HES5 promoter area with the transient overexpression of JAG1 (**p < 0.01 by Student's t test, n = 3).
Conjugate: Green dye

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 1 Effects of catecholamines and inflammatory mediators under normoxia conditions on cEND cells. Cells were grown to confluence with subsequent differentiation. Catecholamines (CAT) or inflammatory mediators (INF) were applied for 24 h under normoxia conditions (24 h NORMOX). (A,B) Immunofluorescence staining of tight junction proteins claudin-5 (green) and ZO-1 (red) as markers of morphological changes of the endothelial cell monolayer. DAPI (blue) was used for staining of nuclei. Magnification 400 times, scale bar 20 mum. (C) Cell number after CAT and INF treatment normalized to control. (D) CAT induced changes of ZO-1 protein expression in cEND cells investigated by Western blot. Data is presented as the means (+- SEM) of n = 5 independent experiments. Altered protein expression was normalized to beta-actin. ZO-1 protein level changes are expressed as fold over untreated control, which was set 1. * P < 0.05, ** P < 0.01, *** P < 0.001.
Conjugate: Green dye

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 2 Loss of morphological integrity of cEND cells exposed to elevated catecholamine levels and inflammatory mediators. Immunofluorescence staining of tight junction proteins claudin-5 (green) and ZO-1 (red) as markers of morphological changes of the endothelial cell monolayer. DAPI (blue) was used for staining nuclei. Cells were grown on cover slips to confluence. After differentiation cEND cells were treated with combination of catecholamines and inflammatory mediators (stress factors). Stress factors were administered under different incubation conditions. Magnification 400 times, scale bar 20 mum. (A) Stress factor application for 4h under normoxia conditions (4 h NORMOX). (B) Stress factor application for 24 h under normoxia conditions (24h NORMOX). (C) . Stress factor application for 4 h under oxygen glucose deprivation conditions (OGD). (D) Stress factor application for 4 h under OGD conditions with 20 h of subsequent reoxygenation under normoxia conditions (REOX). (E) Cell number in treatments shown in (A-D) normalized to control. *** P < 0.001, **** P < 0.0001.
Conjugate: Green dye

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 1. Characterization of mouse RPE/choroid by scRNAseq and identification of transcriptionally distinct choroidal EC subtypes. (a) Scheme of the preparation of RPE/choroid single-cell suspensions by sequential enzymatic digestion and cell sorting. Hoechst + /TO-PRO-3 - live cells were subjected to scRNAseq. (b) Principal-component analysis of 7,723 single-cell expression profiles obtained from RPE/choroid tissue from four mice (C57BL/6J and B6129PF1/J strains, one male and one female per strain). Data are shown in two dimensions using tSNE. Unsupervised analysis clustered cells into 13 transcriptionally distinct cell populations, each plotted in a different color. (c) Identification of cell types in RPE/choroid tissue from B6129PF1/J mice according to the average expression of known markers in each cluster. Cluster number colors are the same as in panel b. Relative gene expression among cell types was calculated using the average normalized UMIs in each cluster and represented as the percentage of the cluster with maximum expression. White, 0%; red, 100%. (d) Genes with at least fivefold differential expression between Kdr high (green bars) and Kdr low (purple bars) ECs (Benjamini-Hochberg adjusted likelihood-ratio test, adj P < 0.05). Only genes with maximum average normalized UMIs >=1 are shown. Relative gene expression in Kdr med ECs (brown bars) is also included. Top: Kdr high expression at least fivefold higher than Kdr low . Bottom, Kdr low expression at least five
Conjugate: Green dye

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig 7 Junction protein expression in fresh and cryopreserved hCMEC/D3 monolayers. The expression of claudin-5 and zonula occludens (ZO)-1 in hCMEC/D3 monolayers that were non-cryopreserved (fresh, unfrozen control), or were loaded with 5% DMSO, 6% HES, and 2% chondroitin sulfate, subjected to cooling at 1degC/min to -40degC, plunging and storage in liquid nitrogen, rapid thawing, and immediate cryoprotectant removal. Fresh and cryopreserved monolayers were stained with the corresponding antibody conjugated to Alexa Fluor 488. The scale bar for claudin-5-stained fresh, unfrozen cells applies to all images.
Conjugate: Green dye

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: 3 Figure F4L5.13 promotes endothelial cell barrier function Expression of endothelial junction protein ZO-1 and stabilization of betacatenin in bEnd.3 cells treated with 30 nM F4L5.13 or 30 nM recombinant NDP for the indicated times. Blots are representative of five different experiments. Histogram represents the ratio of ZO-1 and betacatenin levels over beta-Tubulin measured by densitometry of five independent experiments. Data are presented as mean +- SEM. Significance was calculated by one-way ANOVA with Bonferroni's multiple comparisons test (* P < 0.05 as compared with NT). Immunofluorescence of ZO-1, CLDN3, and CLDN5 localization at bEnd.3 cell junctions. bEnd.3 cells were treated or not with 30 nM F4L5.13 or 30 nM Norrin (NDP) with or without 100 ng/ml VEGF for 1 h. ZO-1 is shown in green, CLDN3/5 in red, and DAPI in blue. Scale bars: 15 um. Quantification of ZO-1, CLDN3, and CLDN5 fluorescence intensity. Each column represents 40 measurements of fluorescence intensity per condition (y-axis). Data are presented as mean +- SEM. Significance was calculated by one-way ANOVA with Bonferroni's multiple comparisons test (* P < 0.05 as compared to VEGF treatment). Transendothelial permeability assay quantifying the passage of FITC-dextran through a monolayer of bEnd.3 cells. Passage of FITC-dextran was measured after exposure of bEnd.3 cells to 100 ng/ml VEGF, 30 nM F4L5.13 or both or pretreated with VEGF for 1 h before treating with F4L5.13 for 1 h. Data are presented as mea
Conjugate: Green dye

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 3 Validation of uE-BBB. A) Excised membranes were stained for cell line specific markers. The bEnd.3 cells were stained for TJ markers, ZO-1 and CLDN-5, after 5 days of growth and the C8-D1A cells were stained for astrocyte marker, GFAP, after 7 days of growth. DRAQ5 was used as a nuclear stain (Biological replicate: n = 1; technical replicates: n = 3). B) Schematic of the uE-BBB lumen channel to the basolateral compartment where resistance of large molecule passage to the basolateral compartment was evaluated after 2, 5, and 7 days of growth. C8-D1A cells were incorporated after day 0 measurements and bEnd.3 cells were incorporated after day 2 measurements. C) Large particle transport assays through uE-BBB were conducted by passing 100 ug mL -1 of 10 kDa FITC-dextran solution through the lumen compartment of the system for 1 h and quantifying the amount of FITC-dextran that had accumulated in the basolateral compartment ( n = 4; Mann-Whitney two-tailed test; * signifies p < 0.05). Initially, there is little resistance to the diffusion of FITC-dextran into the basolateral compartment, but as the device matures, the permeability of the system decreases. D) Utilizing the integrated electrodes of the system, TEER measurements were collected daily. During the first 2 days of culture, the astrocytes alone do not induce any change in resistance. However, the resistance begins to increase following incorporation of bEnd.3 cells after day 2 ( n = 8). All data represented as me
Conjugate: Green dye

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: 4 FIGURE Inhibition of RA signaling disrupts vascular barrier function in the postnatal mouse retina. (A-F) Pups treated with BMS493 or DMSO were systemically injected with sulfo-NHS-biotin 1 h before euthanasia at P4.5. Streptavidin staining reveals leakage of the sulfo-NHS-biotin tracer into retinal tissue of BMS493-treated pups (B) and confinement of sulfo-NHS-biotin to vasculature of control pups (A). (C-F) Extravascular sulfo-NHS-biotin in BMS493-treated pups is apparent in images co-stained for sulfo-NHS-biotin and CLDN5. (G) Quantification of streptavidin fluorescence per retina (DMSO n = 7, BMS493 n = 9). (I-P) Immunostaining for CLDN5, PLVAP, and LEF1 in retinas from P4.5 control and BMS493-treated pups was not remarkably different. (H) Quantification of PLVAP fluorescence (DMSO n = 4, BMS493 n = 5) and LEF1 fluorescence (DMSO n = 7, BMS493 n = 7) normalized to vascular content per field (CLDN5 signal) in the nascent vascular network of BMS493-treated and control pups (average of 2 fields per retina). (Q,R) VEcad immunostaining in retinas of P4.5 BMS493-treated and control pups (representative of DMSO n = 3, BMS493 n = 3; see also Figure S6). Error bars indicate StDev. Significance was assessed by unpaired unequal variance t test (Welch's t test)
Conjugate: Green dye

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 1 Claudin-1 immunoreactivity (IR) and ZO-1 IR are preferentially found in epi-/perineurium in rats' dorsal root ganglia (DRGs), while claudin-19 IR is most abundant in the fiber-rich region. Classification into neuron rich region (NRR, green), NRR epi-/perineurium (EPN, black), fiber rich region (FRR, red), and FRR-EPN (blue) is shown in ( a ). Control DRG sections (CL) from Wistar rats were immunostained. IRs of claudin-1, claudin-5, and ZO-1 were quantified and compared between NRR and FRR, together with their putative EPN. Representative stainings for claudin-1 (red), claudin-5 (green) ( b ), claudin-12 ( c ), claudin-19 ( d ), and ZO-1 ( e ) are shown. Arrows point to structures identified as vessels ( b ), Schwann cells ( c ), and paranodes of Schwann cells ( d ). Quantification of the signal intensity in the specified areas ( f ). (Scale bars = 100 um; n = 5 or 6; claudin-1: NRR versus NRR-EPN, NRR versus FRR-EPN, FRR versus NRR-EPN, and FRR versus FRR-EPN: p < 0.0001; ZO-1: FRR-EPN versus NRR: p = 0.0003; FRR-EPN versus NRR-EPN: p = 0.0138; FRR-EPN versus NRR: p = 0.00018. No normal distribution: ZO-1 FRR. No variance homogeneity: claudin-1. Two-way ANOVA, Tukey HSD. * p < 0.05, *** p < 0.001).
Conjugate: Green dye

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 4 Claudin-1 and ZO-1 IR are unchanged after CCI. DRG sections from Wistar rats with 7 d CCI and naive controls were immunostained. IRs of claudin-1, claudin-5, and ZO-1 were quantified and compared between IL and CL DRGs after CCI in the neuron rich region (NRR) and fiber rich region (FRR), and their putative EPN are presented as light intensity per um 2 . Representative sections of claudin-1 (red) and claudin-5 (green) ( a ), and ZO-1 (green) ( b ) are shown. Quantification of the signal intensity in the specified areas: significant differences between regions were not indicated, as they are already analyzed in Figure 1 ( c ). (Scale bars = 100 um. n = 5 or 6; claudin-1: NRR versus NRR-EPN, NRR versus FRR-EPN, FRR versus NRR-EPN, and FRR versus FRR-EPN: p < 0.0001; ZO-1: FRR-EPN versus NRR: p = 0.0003; FRR-EPN versus NRR-EPN: p = 0.0138; FRR-EPN versus NRR: p = 0.00018. No normal distribution: ZO-1 CL FRR. No variance homogeneity: claudin-1 CL. Two-way ANOVA, Tukey HSD).
Conjugate: Green dye

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 6 Claudin-5 IR within vessels and Cldn5 mRNA in the NRR are reduced in rat DRGs. DRG sections from Wistar rats after CCI were immunostained. Quantification of claudin-5 and van Willebrand factor (vWF) IRs and comparison between IL and CL DRGs after CCI in the NRR and FRR as light intensity per um 2 . Tissue samples from the NRR and vessels of the FRR were obtained using laser microdissection (LMD). Claudin-5 and vWF mRNA were quantified as relative expression (to GAPDH) via RT-qPCR. Representative sections of co-stainings with claudin-5 (green) and vWF (red) ( a ). Quantification of the claudin-5 and vWF IR ( b ) n = 6; claudin-5: FRR versus NRR, p = 0.000261; NRR: CL versus CCI, p = 0.02985; vWF: NRR versus FRR, p = 0.00515. No normal distribution: claudin-5 FRR CCI, vWF NRR CL. No variance homogeneity: claudin-5 FRR. Two-way ANOVA, Tukey HSD.) And quantification of mRNA expression ( c ) n = 5; Cldn5 : NRR versus FRR, p = 0.0136; CL versus IL: p = 0.0448; Vwf : NRR versus FRR, p = 0.00838; two-way ANOVA, Tukey HSD) in the specified areas. (Scale bars = 100 um. * p < 0.05, ** p < 0.01, *** p < 0.001.) All results are summarized in Table 1 .
Conjugate: Green dye

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 4 Abnormal intratumoral blood vessels in AT/RT xenografts. (A) Confocal microscopy images of a sagittal section of the brain of a mouse carrying a VUMC-AT/RT-01 xenograft, stained for the endothelial cell marker CD31, showing reduced density but increased diameter of intratumoral blood vessels. (B) Confocal microscopy image of the brain of a mouse carrying a VUMC-AT/RT-01 xenograft, stained for the blood-brain barrier endothelial cell markers GLUT1 and CLDN5, showing reduced GLUT1 expression in intratumoral blood vessels. (C) Confocal microscopy image of the brain of a mouse carrying a VUMC-AT/RT-01 xenograft, stained for the tight junction protein CLDN5, showing disorganization of tight junctions between endothelial cells of intratumoral blood vessels. (D) Quantification of vessel density, as determined by area of tissue staining positive for CD31, in VUMC-AT/RT-01 xenografts compared with normal brain. *** P < 0.001 (Mann-Whitney U -test). (E) Quantification of vessel diameter of intratumoral blood vessels in VUMC-AT/RT-01 xenografts compared with blood vessels in normal brain tissue. ** P < 0.01, **** P < 0.0001 (Dunnett's multiple comparisons test).
Conjugate: Green dye

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 2. Dlg1 genetically interacts with Fz4 to regulate retinal angiogenesis and the BRB. ( A-B ) Maximum projection of the superficial, intermediate, and deep vascular plexuses of P18 retinas from the indicated genotypes (column 1), with boxed regions displayed at higher magnification in columns 2-5. Mice were injected IP with 2-3 mg of sulfo-NHS-biotin 1-2 hr before sacrifice. Scale bar for column 1, 400 mum. Scale bar for columns 2-5, 200 mum. ( C-E ) Quantification of vascular density in the superficial and deep plexuses ( C ), the fraction of vessels that immunostain for Claudin5 ( D ), and the fraction of vessels that immunostain for PLVAP ( E ), for the genotypes shown in ( A ), ( B ), and Figure 1J . In this Figure and Figures 3 and 4 , the numbers of WT alleles for the genes of interest are listed below each set of data points.
Conjugate: Green dye

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 8. Mutation of the Fz4 C-terminal PDZ-binding domain enhances the severity of retinal vascular defects in a Dlg1 mutant background. ( A ) Diagram of the Fz4 allele produced by CRISPR/Cas9 editing ( Fz4 MGK ). Numbers indicate exons. TVV, the C-terminal three amino acids, were substituted with MGK. Right, sequencing chromatograms from mouse tail PCR products. Boxes encompass the three altered codons. Top, WT allele. Bottom, Fz4 MGK allele. ( B ) Protein extracts from HEK/293T cells transfected with WT Myc-Dlg1 (+) or an empty expression vector (-) were subjected to affinity capture using a biotin-Fz4 WT C-terminal peptide, a biotin-Fz4 peptide with the C-terminal three amino acids substituted by MGK, or a biotin-rhodopsin C-terminal peptide. Myc-Dlg1 binds only to the Fz4 WT C-terminal peptide. ( C ) Immunoblot of protein extracts from HEK/293T cells transfected with Rim-Fz4 WT, Rim-Fz4(4A) or Rim-Fz4(MGK), i.e., Fz4 with the C-terminal three amino acids changed to MGK. ( D-F ) Maximum projection of the superficial, intermediate, and deep vascular plexuses of P18 retinas from the indicated genotypes (column 1) with boxed regions enlarged in columns 2-5. Mice were injected IP with 2-3 mg of sulfo-NHS-biotin 1-2 hr before sacrifice. Arrow in ( E ) indicates biotin leakage. Scale bar for column 1, 400 mum. Scale bar for columns 2-5, 200 mum. ( G-I ) Quantification of summed vascular density ( G ), the fraction of vessels that immunostain for Claudin5 ( H ), and the
Conjugate: Green dye

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 1: Meningeal lymphatic sub-arachnoid extensions uptake molecules and immune cells from the cerebrospinal fluid. a, General scheme of meningeal lymphatic vascular organization. b, Scheme of the experiment presented in (c). Prox1 GFP mice were injected into the cisterna magna (i.c.m.) with 5ul of Qdot 655 . The transverse sinus was imaged through a thinned skull. c, Representative image of meningeal lymphatics adjacent to the transverse sinus (Prox1 GFP - green) filled with the i.c.m. injected Qdot 655 (red) 60min after injection. The inset represents the coronal view of the lymphatic vessel filled with Qdot 655 . Scale bar = 65 um. Representative of 3 independent animals. d, Representative images of the lymphatic vessels co-labeled with i.c.m. injected anti-Lyve-1 A488 and exogenously applied anti-Lyve-1 A660 at different time points after i.c.m. injection. Arrows in inset at 5 and 15 min illustrate the initial points where the i.c.m. injected anti-Lyve-1 A488 labelled the meningeal lymphatics. Scale bar = 1000 um (upper panel); 250 um (bottom left panel) 60 um (bottom right panel). e, Quantification of the percentage of lymphatic vessels labeled by the i.c.m. injected antibody and total lymphatic area at different time points post injection (mean +- s.e.m.; n=4 mice/group). f, Representative images of OVA 594 and fluorescent bead accumulation along the lymphatics (Lyve-1 - red) are shown. Arrows point to lymphatic extensions in the area of microbeads
Conjugate: Green dye

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 8. S1PR1/ss-arrestin coupling in aorta-associated lymphatic vessels and S1P-dependent signaling in LECs. ( A ) Representative confocal image of a sagittal cryosection (14 uM) of a S1RP1-GS mouse aorta immunostained for VEC and lymphatic vessel endothelial hyaluronic acid receptor 1 (LYVE1) (N = 2). White arrows indicate GFP+ arterial ECs at a branch point orifices and yellow arrows indicate adventitia-associated GFP+ LECs. Scale bar is 50 uM. ( B ) Confocal image of a S1RP1-GS mouse thoracic aorta whole-mount preparation immunostained for vascular endothelial growth factor receptor 3 (VEGFR3; Flt4 ), LYVE1, and CLDN5 with the tunica intima in contact with coverslip. Orange stars indicate VEGFR3+LYVE1+GFP+ areas, cyan stars indicate VEGFR3+LYVE1 low GFP+ areas, and magenta stars indicate VEGFR3+LYVE1+GFP- areas. Image is representative of observations from three mice (see also Figure 8--figure supplement 1B ). Scale bar is 100 uM. ( C-D ) Representative confocal images of mesenteric lymphatics from H2B-GFP control ( C ) and S1PR1-GS mice bearing Sphk2 -/- : Sphk1 f/f (Cre-) or Sphk2 -/- : Sphk1 f/f : Lyve1-Cre (Lyve1-Cre+) alleles whole-mounted and immunostained for PROX1 and ASMA. White arrows indicate ASMA+PROX1+GFP+, yellow arrows indicate PROX1+GFP-, cyan arrows indicate ASMA+PROX1+GFP-. Scale bars are 100 uM. ( E ) Quantification of GFP+ signal over ASMA+PROX1+ and ASMA-PROX1+ areas from Cre- (n = 5), Lyve1-Cre (n = 4), and H2B-GFP control (n = 2) mice. One-way ANO
Conjugate: Green dye