anti lyve1 antibody  (Cell Signaling Technology Inc)

Journal: Translational Stroke Research

Article Title: CircRNA circ_0004058 Modulates Early Brain Injury in Subarachnoid Hemorrhage Through miR-221-3p and VE1 Activation Pathway

doi: 10.1007/s12975-025-01383-9

Figure Lengend Snippet: Identification of key genes involved in SAH onset and validation in vivo. A : Volcano plot of differentially expressed genes between samples of brain tissue from SAH (n = 10) and sham (n = 10) rats according to RNA sequencing; red represents upregulated genes and green represents downregulated genes. B : Venn diagram of differentially expressed genes according to RNA sequencing and SAH-related genes analyzed by GeneCards. C–D : Expression levels of LYVE1 (C) and TEK (D) extracted from RNA sequencing data. E–F : mRNA and protein levels of LYVE1 in the hippocampal tissue of sham-operated and SAH rats, as detected by RT-qPCR (E) and Western blotting (F). G : Flow cytometry detection of LYVE1 + CD68 + macrophages in the hippocampal tissue of sham-operated and SAH rats. H–I : mRNA and protein levels of VEGF-A in the hippocampal tissue of sham-operated and SAH rats, as detected by RT-qPCR (H) and Western blotting (I). (J) Immunohistochemistry detection of CD31 and VEGFA protein levels in the hippocampal tissue of each group of rats. n = 8 rats per treatment. *** p < 0.001 vs. sham-operated rats

Article Snippet: After blocking with 5% bovine serum albumin, membranes were incubated overnight at 4 °C with rabbit monoclonal antibodies against LYVE1 (1:1000, #67,538, Cell Signaling Technology), vascular endothelial growth factor (VEGF)-A (1:1000, ab214424, Abcam), and GAPDH (ab181602, 1:10,000, Abcam).

Techniques: Biomarker Discovery, In Vivo, RNA Sequencing, Expressing, Quantitative RT-PCR, Western Blot, Flow Cytometry, Immunohistochemistry

Journal: Translational Stroke Research

Article Title: CircRNA circ_0004058 Modulates Early Brain Injury in Subarachnoid Hemorrhage Through miR-221-3p and VE1 Activation Pathway

doi: 10.1007/s12975-025-01383-9

Figure Lengend Snippet: Effects of LYVE1 overexpression on brain injury in SAH rats. ote: SAH rats were treated with oe-NC or oe-LYVE1. A : RT-qPCR detection of LYVE1 mRNA expression in the hippocampal tissue of SAH rats. B : Western blot of LYVE1 protein expression in the hippocampal tissue of SAH rats. C–D : Neurological function scores (C) and extent of cerebral edema (D) in SAH rats. E : The pathological scores of SAH rats. F : Representative images of brain specimens collected after SAH induction and quantitative assessment of SAH severity; G : Evans blue staining was used to evaluate BBB permeability and quantify Evans blue dye extravasation in each group; scale bar = 0.5 cm; H : Nissl staining of neuronal injury in the hippocampal tissue of SAH rats. I : TUNEL staining of neuronal apoptosis in the hippocampal tissue of SAH rats. J : Flow cytometry analysis of LYVE1 + CD68 + macrophages in the hippocampal tissue of SAH rats. K–L : mRNA and protein levels of VEGF-A in the hippocampal tissue of SAH rats, as detected by RT-qPCR (K) and Western blotting (L); M: Immunohistochemistry was used to detect CD31 and VEGFA protein levels in the hippocampal tissue of each group. n = 8 rats per treatment. ** p < 0.01, *** p < 0.001vs. treatment with oe-N

Article Snippet: After blocking with 5% bovine serum albumin, membranes were incubated overnight at 4 °C with rabbit monoclonal antibodies against LYVE1 (1:1000, #67,538, Cell Signaling Technology), vascular endothelial growth factor (VEGF)-A (1:1000, ab214424, Abcam), and GAPDH (ab181602, 1:10,000, Abcam).

Techniques: Over Expression, Quantitative RT-PCR, Expressing, Western Blot, Staining, Permeability, TUNEL Assay, Flow Cytometry, Immunohistochemistry

Journal: Translational Stroke Research

Article Title: CircRNA circ_0004058 Modulates Early Brain Injury in Subarachnoid Hemorrhage Through miR-221-3p and VE1 Activation Pathway

doi: 10.1007/s12975-025-01383-9

Figure Lengend Snippet: Identification of miRNAs upstream of LYVE1. A : Volcano plot of differentially expressed miRNAs between control (n = 2) and SAH (n = 2) samples in the GSE161870 dataset. B: Venn diagram of regulatory miRNAs upstream of LYVE1, as predicted by miRWalk database, and differentially expressed miRNAs in the GSE161870 dataset. C : Heat map of the expression of candidate miRNAs in the GSE161870 dataset. D : miR-221-3p expression in the hippocampal tissue of sham-operated and SAH rats, as detected by RT-qPCR. E : miR-221-3p binding sites on LYVE1 mRNA, as predicted by miRWalk database. F : miR-221-3p binding to LYVE1, as confirmed by dual-luciferase reporter assay. G : Expression levels of miR-221-3p and LYVE1 in RMA-BMs co-cultured with primary rat hippocampal neurons that had been transfected with miR-221-3p mimic or miR-221-3p inhibitor and then treated with GW4869, as detected by RT-qPCR. H : Western blot of LYVE1 protein expression in RMA-BMs co-cultured with primary rat hippocampal neurons that had been transfected with miR-221-3p mimic or miR-221-3p inhibitor and then treated with GW4869. n = 8 rats per treatment. *** p < 0.001 vs. treatment with NC mimic or NC inhibitor. Cell experiments were repeated three times

Article Snippet: After blocking with 5% bovine serum albumin, membranes were incubated overnight at 4 °C with rabbit monoclonal antibodies against LYVE1 (1:1000, #67,538, Cell Signaling Technology), vascular endothelial growth factor (VEGF)-A (1:1000, ab214424, Abcam), and GAPDH (ab181602, 1:10,000, Abcam).

Techniques: Control, Expressing, Quantitative RT-PCR, Binding Assay, Luciferase, Reporter Assay, Cell Culture, Transfection, Western Blot

Journal: Translational Stroke Research

Article Title: CircRNA circ_0004058 Modulates Early Brain Injury in Subarachnoid Hemorrhage Through miR-221-3p and VE1 Activation Pathway

doi: 10.1007/s12975-025-01383-9

Figure Lengend Snippet: Effects of miR-221-3p-mediated regulation of LYVE1 on EBI in SAH rats. Note: SAH rats were treated with miR-221-3p inhibitor alone or in combination with sh-LYVE1. A : RT-qPCR detection of miR-221-3p expression and LYVE1 mRNA expression in the hippocampal tissue of SAH rats. B : Western blot of LYVE1 protein expression in the hippocampal tissue of SAH rats. C – D : Neurological function scores (C) and extent of cerebral edema (D) in SAH rats. E : The pathological scores of SAH rats; F : Representative images of brain specimens collected after SAH induction and quantitative assessment of SAH severity. G : Evans blue staining to assess BBB permeability and quantify Evans blue dye extravasation in each group; scale bar = 0.5 cm; H : Nissl staining of neuronal injury in the hippocampal tissue of SAH rats. I : TUNEL staining of neuronal apoptosis in the hippocampal tissue of SAH rats. J : Flow cytometry analysis of LYVE1 + CD68. + macrophages in the hippocampal tissue of SAH rats. K–L : mRNA and protein levels of VEGF-A in the hippocampal tissue of SAH rats, as detected by RT-qPCR (K) and Western blotting (L). M : Immunohistochemistry to detect CD31 and VEGFA protein levels in the hippocampal tissue of each group. n = 8 rats per treatment. *** p < 0.001

Article Snippet: After blocking with 5% bovine serum albumin, membranes were incubated overnight at 4 °C with rabbit monoclonal antibodies against LYVE1 (1:1000, #67,538, Cell Signaling Technology), vascular endothelial growth factor (VEGF)-A (1:1000, ab214424, Abcam), and GAPDH (ab181602, 1:10,000, Abcam).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Staining, Permeability, TUNEL Assay, Flow Cytometry, Immunohistochemistry

Journal: Translational Stroke Research

Article Title: CircRNA circ_0004058 Modulates Early Brain Injury in Subarachnoid Hemorrhage Through miR-221-3p and VE1 Activation Pathway

doi: 10.1007/s12975-025-01383-9

Figure Lengend Snippet: Relationship among circ_0004058, miR-221-3p, and LYVE1. A : Volcano plot of differentially expressed circRNAs between control (n = 4) and SAH (n = 5) samples in the GSE161913 dataset. B : Venn diagram of regulatory circRNAs upstream of miR-221-3p, as predicted by circBank database, and differentially expressed circRNAs in the GSE161913 dataset. C : Differential expression analysis of circ_0000826 and circ_0004058 in the GSE161913 dataset (Control, n = 4; SAH, n = 5). D : Stability of circ_0004058, as determined by RNase R digestion. E : circ_0004058 expression in the hippocampal tissue of sham-operated and SAH rats, as detected by RT-qPCR. F : circ_0004058 and miR-221-3p binding sites, as predicted by starBase database. G : Binding of circ_0004058 to miR-221-3p, as confirmed by dual-luciferase reporter assay. H : circ_0004058 binding to miR-221-3p, as detected by RNA pull-down assays. I : circ_0004058 binding to miR-221-3p, assessed by RIP assays. J : Localization of circ_0004058 and miR-221-3p in primary rat hippocampal neurons, as detected by FISH assays. K : Expression levels of miR-221-3p and LYVE1 in RMA-BMs co-cultured with primary rat hippocampal neurons that had been transfected with oe-circ_0004058 or sh-circ_0004058 and then treated with GW4869, as detected by RT-qPCR. L : Western blot of LYVE1 protein expression in RMA-BMs co-cultured with primary rat hippocampal neurons that had been transfected with oe-circ_0004058 or sh-circ_0004058 and then treated with GW4869. n = 8 rats per treatment. ** p < 0.01, *** p < 0.001 vs. treatment with oe-NC or sh-NC. Cell experiments were repeated three times

Article Snippet: After blocking with 5% bovine serum albumin, membranes were incubated overnight at 4 °C with rabbit monoclonal antibodies against LYVE1 (1:1000, #67,538, Cell Signaling Technology), vascular endothelial growth factor (VEGF)-A (1:1000, ab214424, Abcam), and GAPDH (ab181602, 1:10,000, Abcam).

Techniques: Control, Quantitative Proteomics, Expressing, Quantitative RT-PCR, Binding Assay, Luciferase, Reporter Assay, Cell Culture, Transfection, Western Blot

Journal: Translational Stroke Research

Article Title: CircRNA circ_0004058 Modulates Early Brain Injury in Subarachnoid Hemorrhage Through miR-221-3p and VE1 Activation Pathway

doi: 10.1007/s12975-025-01383-9

Figure Lengend Snippet: Effects of circ_0004058-mediated regulation of miR-221-3p and reduced EBI in SAH rats. Note: SAH rats were treated with oe-circ_0004058 alone or in combination with miR-221-3p mimic. A: RT-qPCR detection of circ_0004058, miR-221-3p, and LYVE1 expression in the hippocampal tissue of SAH rats. B: Western blot of LYVE1 protein expression in the hippocampal tissue of SAH rats. C–D: Neurological function scores (C) and extent of cerebral edema (D) in SAH rats. E: The pathological scores of SAH rats. F: Representative images of brain specimens collected after SAH induction and quantitative assessment of SAH severity. G: Evans blue staining to evaluate BBB permeability and quantitative analysis of Evans blue dye extravasation in each group; scale bar = 0.5 cm. H: Nissl staining of neuronal injury in the hippocampal tissue of SAH rats. I: TUNEL staining of neuronal apoptosis in the hippocampal tissue of SAH rats. J: Flow cytometry analysis of LYVE1 + CD68 + macrophages in the hippocampal tissue of SAH rats. K–L: mRNA and protein levels of VEGF-A in the hippocampal tissue of SAH rats, as detected by RT-qPCR (K) and Western blotting (L). M: Immunohistochemistry to detect CD31 and VEGFA protein levels in the hippocampal tissue of each group. n = 8 rats per treatment. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. treatment with oe-NC + NC mimic or oe-circ_0004058 + NC mimic

Article Snippet: After blocking with 5% bovine serum albumin, membranes were incubated overnight at 4 °C with rabbit monoclonal antibodies against LYVE1 (1:1000, #67,538, Cell Signaling Technology), vascular endothelial growth factor (VEGF)-A (1:1000, ab214424, Abcam), and GAPDH (ab181602, 1:10,000, Abcam).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Staining, Permeability, TUNEL Assay, Flow Cytometry, Immunohistochemistry

Journal: Translational Stroke Research

Article Title: CircRNA circ_0004058 Modulates Early Brain Injury in Subarachnoid Hemorrhage Through miR-221-3p and VE1 Activation Pathway

doi: 10.1007/s12975-025-01383-9

Figure Lengend Snippet: Effects of the circ_0004058/miR-221-3p/LYVE1 axis on EBI in SAH rats. Note: SAH rats were treated with oe-circ_0004058 alone or in combination with sh-LYVE1. A: RT-qPCR detection of circ_0004058, miR-221-3p, and LYVE1 expression in the hippocampal tissue of SAH rats. B: Western blot of LYVE1 protein expression in the hippocampal tissue of SAH rats. C–D: Neurological function scores (C) and extent of cerebral edema (D) in SAH rats. E: The pathological scores of SAH rats. F: Representative images of brain specimens collected after SAH induction and quantitative assessment of SAH severity. G: Evans blue staining to evaluate BBB permeability and quantitative analysis of Evans blue dye extravasation in each group; scale bar = 0.5 cm. H: Nissl staining of neuronal injury in the hippocampal tissue of SAH rats. I: TUNEL staining of neuronal apoptosis in the hippocampal tissue of SAH rats. J: Flow cytometry analysis of LYVE1 + CD68 + macrophages in the hippocampal tissue of SAH rats. K–L: mRNA and protein levels of VEGF-A in the hippocampal tissue of SAH rats, as detected by RT-qPCR (K) and Western blotting (L). M: Immunohistochemistry to detect CD31 and VEGFA protein levels in the hippocampal tissue of each group. n = 8 rats per treatment. *** p < 0.001 vs. treatment with oe-NC + sh-NC; # p < 0.05 vs. treatment with oe-circ_0004058 + sh-NC

Article Snippet: After blocking with 5% bovine serum albumin, membranes were incubated overnight at 4 °C with rabbit monoclonal antibodies against LYVE1 (1:1000, #67,538, Cell Signaling Technology), vascular endothelial growth factor (VEGF)-A (1:1000, ab214424, Abcam), and GAPDH (ab181602, 1:10,000, Abcam).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Staining, Permeability, TUNEL Assay, Flow Cytometry, Immunohistochemistry

Journal: Translational Stroke Research

Article Title: CircRNA circ_0004058 Modulates Early Brain Injury in Subarachnoid Hemorrhage Through miR-221-3p and VE1 Activation Pathway

doi: 10.1007/s12975-025-01383-9

Figure Lengend Snippet: Effects of LYVE1 + CD68 + macrophages on EBI in SAH rats. Note: SAH rats were treated with oe-LYVE1 alone or in combination with anti-CSF1R antibody. A: RT-qPCR detection of circ_0004058, miR-221-3p, and LYVE1 expression in the hippocampal tissue of SAH rats. B: Western blot of LYVE1 protein expression in the hippocampal tissue of SAH rats. C–D: Neurological function scores (C) and extent of cerebral edema (D) in SAH rats. E: The pathological scores of SAH rats. F: Representative images of brain specimens collected after SAH induction and quantitative assessment of SAH severity. G: Evans blue staining to assess BBB permeability and quantitative analysis of Evans blue dye extravasation in each group; scale bar = 0.5 cm. H: Nissl staining of neuronal injury in the hippocampal tissue of SAH rats. I: TUNEL staining of neuronal apoptosis in the hippocampal tissue of SAH rats. J: Flow cytometry analysis of LYVE1 + CD68 + macrophages in the hippocampal tissue of SAH rats. K–L: mRNA and protein levels of VEGF-A in the hippocampal tissue of SAH rats, as detected by RT-qPCR (K) and Western blotting (L). n = 8 rats per treatment. M: Immunohistochemistry to detect CD31 and VEGFA protein levels in the hippocampal tissue of each group. ** p < 0.01, *** p < 0.001 vs. treatment with oe-NC + anti-IgG; # p < 0.05 vs. treatment with oe-LYVE1 + anti-IgG

Article Snippet: After blocking with 5% bovine serum albumin, membranes were incubated overnight at 4 °C with rabbit monoclonal antibodies against LYVE1 (1:1000, #67,538, Cell Signaling Technology), vascular endothelial growth factor (VEGF)-A (1:1000, ab214424, Abcam), and GAPDH (ab181602, 1:10,000, Abcam).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Staining, Permeability, TUNEL Assay, Flow Cytometry, Immunohistochemistry

Journal: Translational Stroke Research

Article Title: CircRNA circ_0004058 Modulates Early Brain Injury in Subarachnoid Hemorrhage Through miR-221-3p and VE1 Activation Pathway

doi: 10.1007/s12975-025-01383-9

Figure Lengend Snippet: Schematic diagram of the mechanism by which the circ_0004058/miR-221-3p/LYVE1 axis affects SAH-induced EBI

Article Snippet: After blocking with 5% bovine serum albumin, membranes were incubated overnight at 4 °C with rabbit monoclonal antibodies against LYVE1 (1:1000, #67,538, Cell Signaling Technology), vascular endothelial growth factor (VEGF)-A (1:1000, ab214424, Abcam), and GAPDH (ab181602, 1:10,000, Abcam).

Techniques:

Journal: Nature

Article Title: Increased CSF drainage by non-invasive manipulation of cervical lymphatics

doi: 10.1038/s41586-025-09052-5

Figure Lengend Snippet: a , Sequence of intracisternal (i.c.) infusion of FluoSpheres into Macaca fascicularis monkeys at 2.5 ml over 10 min followed by measurement of FluoSpheres in cervical lymph nodes and hard palate at 180 min. Before infusion, 1 ml of CSF was removed at the cisterna magna over 10 min. Anesthesia was administered until the end of the infusion and then readministered at 180 min. b , Immunofluorescence images showing the distribution of FluoSpheres (red) and LYVE1 + vessels in submandibular, parotid, and retropharyngeal lymph nodes (LN). White dashed line boxes mark regions enlarged in panels below. Red arrowheads mark regions with abundant FluoSpheres in lymph node medullary sinusoids. Scale bars, 1 mm. Representative of n = 2 monkeys from two independent experiments. c , Fluorescence image showing the distribution of FluoSpheres (red) in the right-side of the hard palate of a Macaca fascicularis monkey. FluoSpheres (red arrowheads) are abundant near the incisive and greater palatine foramina (white dashed ellipses) in the hard palate. Scale bar, 5 mm. Representative of n = 2 monkeys from two independent experiments. Anatomical positions are indicated in the top right corner: A, anterior; P, posterior; M, medial; L, lateral.

Article Snippet: Primary antibodies used were: anti-mouse LYVE1 (rabbit polyclonal; 11-034, Angiobio); anti-mouse VEGFR3 (goat polyclonal; AF743, R&D Systems); anti-mouse αSMA-Cy3 (mouse monoclonal, clone 1A4; C6198, Sigma); anti-mouse laminin α5 (rabbit polyclonal; EWL004, kerafast); anti-mouse CD31 (hamster monoclonal, clone 2H8; MAB1398Z, Merck); anti-mouse tyrosine hydroxylase (rabbit polyclonal; AB152, Merck); anti-mouse vesicular acetylcholine transporter (VAChT, also known as solute carrier family 18 (vesicular acetylcholine), member 3, Slc18a3, goat polyclonal; ABN100, Merck); anti-monkey LYVE1 (rabbit polyclonal; DP3500, OriGene); anti-mouse eNOS antibody (rabbit polyclonal; ab5589, Abcam); anti-mouse phospho-eNOS antibody (rabbit polyclonal; 9571, Cell Signaling); anti-mouse FOXP2 antibody (goat polyclonal; ab1307, Abcam); anti-mouse ER-TR7 antibody (rat monoclonal, clone ER-TR7; sc-73355, Santa Cruz Biotechnology); anti-mouse Col1a1 (rabbit monoclonal, clone E8F4L; 72026, Cell Signaling); and anti-mouse PDGFRα (goat polyclonal; AF1062, R&D Systems).

Techniques: Sequencing, Immunofluorescence, Fluorescence

Journal: Nature

Article Title: Increased CSF drainage by non-invasive manipulation of cervical lymphatics

doi: 10.1038/s41586-025-09052-5

Figure Lengend Snippet: a , Sequence of intracisternal (i.c.) infusion of 1.0 μl FluoSpheres over 1 min into Prox1 -GFP mice followed by imaging of FluoSphere distribution in the nasal cavity and hard palate 60 min later. b , Brightfield microscopic image showing the anatomical location of the incisive foramen (blue dashed elliptical circle), greater palatine foramen (red dashed circle), and greater palatine nerve (GPN, red dashed lines). The black dashed line marks the border of the olfactory epithelium. Scale bar, 1 mm. Representative of n = 3 mice from three independent experiments. c , Brightfield, fluorescence, and immunofluorescence images of whole mounts showing connections between lymphatics in the nasal cavity and hard palate through the incisive foramen. Brightfield image showing the anatomical location of the incisive foramen (blue dashed elliptical circle). White dashed line box is enlarged in brightfield and fluorescence images in the right two panels that show connections between lymphatics in the nasal mucosa and hard palate through the incisive foramen. The black dashed line marks the border of the intracranial olfactory bulb. Scale bar, 1 mm. Representative of n = 4 mice from three independent experiments. d , Immunofluorescence image showing FluoSpheres (red) within lymphatics in the nasal mucosa and hard palate (green arrowheads). The orange dashed line marks the boundary between lymphatics in the nasal mucosa and hard palate. Scale bar, 200 μm. Representative of n = 4 mice from three independent experiments. e , Immunofluorescence image of whole mount showing FluoSpheres (red) and lymphatics ( Prox1 -GFP, green; LYVE1, blue) in the periorbital area and hard palate. FluoSphere fluorescence is strong in lymphatics along the pterygopalatine artery, infraorbital artery in the orbital fissure, greater palatine artery in the greater palatine canal, and hard palate (green arrowheads). Red dashed lines mark the lymphatic pathway from the orbital fissure to periorbital lymphatics. White dashed lines mark the lymphatic pathway from descending branch of pterygopalatine artery through the greater palatine canal to the hard palate plexus. Scale bar, 1 mm. Representative of n = 4 mice from three independent experiments. f , Immunofluorescence image of coronal section of the greater palatine canal showing FluoSpheres (red) in lymphatics (green) along the pterygopalatine artery, in the greater palatine canal, and in the nasopharyngeal lymphatic plexus (green arrowheads). The white dashed lines mark the lymphatic pathway from the greater palatine canal to the hard palate. Scale bar, 500 μm. Representative of n = 4 mice from three independent experiments. g , Drawing of two lymphatic routes for CSF to reach the hard palate lymphatic plexus, superficial cervical lymphatic scLV-2, and submandibular lymph node (smLN): (1) Meningeal lymphatics that cross the cribriform plate join nasal lymphatics and then traverse the incisive foramen to join the hard palate plexus. (2) Meningeal lymphatics along the pterygopalatine artery travel with the greater palatine artery through the greater palatine canal and join the hard palate lymphatic plexus. Lymphatics in the hard palate plexus carry CSF to scLV-2 en route to the submandibular lymph node. Anatomical positions are indicated in the top right corner: S, superior; I, inferior; A, anterior; P, posterior; M, medial; L, lateral.

Article Snippet: Primary antibodies used were: anti-mouse LYVE1 (rabbit polyclonal; 11-034, Angiobio); anti-mouse VEGFR3 (goat polyclonal; AF743, R&D Systems); anti-mouse αSMA-Cy3 (mouse monoclonal, clone 1A4; C6198, Sigma); anti-mouse laminin α5 (rabbit polyclonal; EWL004, kerafast); anti-mouse CD31 (hamster monoclonal, clone 2H8; MAB1398Z, Merck); anti-mouse tyrosine hydroxylase (rabbit polyclonal; AB152, Merck); anti-mouse vesicular acetylcholine transporter (VAChT, also known as solute carrier family 18 (vesicular acetylcholine), member 3, Slc18a3, goat polyclonal; ABN100, Merck); anti-monkey LYVE1 (rabbit polyclonal; DP3500, OriGene); anti-mouse eNOS antibody (rabbit polyclonal; ab5589, Abcam); anti-mouse phospho-eNOS antibody (rabbit polyclonal; 9571, Cell Signaling); anti-mouse FOXP2 antibody (goat polyclonal; ab1307, Abcam); anti-mouse ER-TR7 antibody (rat monoclonal, clone ER-TR7; sc-73355, Santa Cruz Biotechnology); anti-mouse Col1a1 (rabbit monoclonal, clone E8F4L; 72026, Cell Signaling); and anti-mouse PDGFRα (goat polyclonal; AF1062, R&D Systems).

Techniques: Sequencing, Imaging, Fluorescence, Immunofluorescence

Journal: Nature

Article Title: Increased CSF drainage by non-invasive manipulation of cervical lymphatics

doi: 10.1038/s41586-025-09052-5

Figure Lengend Snippet: a , Sequence of intracisternal (i.c.) infusion of 1.0 μl TMR-dextran or FluoSpheres over 1 min into Prox1 -GFP mice followed by analysis of the distribution of the tracer in the hard palate lymphatic plexus at 60 min. b-d , Fluorescence images showing the distribution of TMR-dextran or FluoSpheres (red) in whole mounts of the hard palate and soft palate of Prox1 -GFP mice ( b , c ). White dashed lines mark border between hard palate and soft palate ( b ). Tracer fluorescence (red) is strong in hard palate lymphatic plexus (red arrowheads) but not in soft palate lymphatic plexus ( b , c white empty arrowheads). Orange arrowheads mark the faint TMR-dextran fluorescence in the nasopharyngeal lymphatic plexus ( b ). Hard palate lymphatic plexus of Prox1 -GFP mouse stained for VEGFR3 (red) and LYVE1 (blue) ( d ). Lymphatic valves ( d , white arrowheads). Initial lymphatics ( d , blue arrowheads) are distributed in the medial and lateral sides of hard palate. Scale bars, 1 mm. Representative of n = 5 mice from three independent experiments. e,f , Images showing TMR-dextran fluorescence (red arrowheads) in the buccal ( e ) and mandibular ( f ) portions of superficial cervical lymphatic scLV-2 between the hard palate lymphatic plexus and submandibular lymph node. White dashed lines ( e ) outline border of hard palate, tongue, and buccal portion of scLV-2. Yellow arrows mark the direction of CSF outflow. Scale bars, 1 mm. Representative of n = 5 mice from three independent experiments. g , Drawing of downstream connection of hard palate plexus to submandibular lymph node (smLN) through buccal and mandibular portions of scLV-2 for CSF drainage. Locations of scLV-1 and scLV-3 are shown for comparison. Left (Lt.), Right (Rt.). Anatomical positions are indicated in the bottom left corner: A, anterior; P, posterior; M, medial; L, lateral.

Article Snippet: Primary antibodies used were: anti-mouse LYVE1 (rabbit polyclonal; 11-034, Angiobio); anti-mouse VEGFR3 (goat polyclonal; AF743, R&D Systems); anti-mouse αSMA-Cy3 (mouse monoclonal, clone 1A4; C6198, Sigma); anti-mouse laminin α5 (rabbit polyclonal; EWL004, kerafast); anti-mouse CD31 (hamster monoclonal, clone 2H8; MAB1398Z, Merck); anti-mouse tyrosine hydroxylase (rabbit polyclonal; AB152, Merck); anti-mouse vesicular acetylcholine transporter (VAChT, also known as solute carrier family 18 (vesicular acetylcholine), member 3, Slc18a3, goat polyclonal; ABN100, Merck); anti-monkey LYVE1 (rabbit polyclonal; DP3500, OriGene); anti-mouse eNOS antibody (rabbit polyclonal; ab5589, Abcam); anti-mouse phospho-eNOS antibody (rabbit polyclonal; 9571, Cell Signaling); anti-mouse FOXP2 antibody (goat polyclonal; ab1307, Abcam); anti-mouse ER-TR7 antibody (rat monoclonal, clone ER-TR7; sc-73355, Santa Cruz Biotechnology); anti-mouse Col1a1 (rabbit monoclonal, clone E8F4L; 72026, Cell Signaling); and anti-mouse PDGFRα (goat polyclonal; AF1062, R&D Systems).

Techniques: Sequencing, Fluorescence, Staining, Comparison

Journal: Nature

Article Title: Increased CSF drainage by non-invasive manipulation of cervical lymphatics

doi: 10.1038/s41586-025-09052-5

Figure Lengend Snippet: a , Immunofluorescence images of whole mounts comparing hard palate lymphatics in adult (8 weeks of age) and aged (90 weeks of age) Prox1 –GFP mice. Like lymphatics, venous sinusoids are PROX1 + . Ageing-related reductions in the lymphatic plexus are outlined by white dashed line boxes that mark regions of interest (ROIs) near the greater palatine nerve (ROI-1) and incisive foramen (ROI-2). Scale bars, 500 μm. Representative of n = 9 mice (adult) and n = 8 mice (aged) from three independent experiments. b , Comparison of lymphatic diameter, VEGFR3 + lymphatic area, LYVE1 intensity and number of lymphatic valves in ROI-1 and ROI-2 in adult (8–10 weeks of age; n = 9) and aged (86–95 weeks of age; n = 8) Prox1 –GFP mice. Each dot is the value for one mouse. The error bars indicate mean ± s.e.m. P values were calculated by two-tailed Welch’s t -test. c , Immunofluorescence images of whole mounts comparing nasal lymphatics in adult (8 weeks of age) and aged (90 weeks of age) Prox1 –GFP mice. Staining as in panel a . ROI-3 marks the measured region of PROX1 + /VEGFR3 + nasal lymphatics (red) for data in panel d . Unlike PROX1 + /VEGFR3 + nasal lymphatics, which are abundant in young adults but less in aged mice, PROX1 + venous sinusoids (green; marked by green arrows) are more abundant in aged mice, as previously described . Scale bars, 500 μm. Representative of n = 4 mice (adult) and n = 4 mice (aged) from three independent experiments. d , Comparison of lymphatic diameter and PROX1 + /VEGFR3 + lymphatic area in nasal lymphatics of adult (8–10 weeks of age; n = 4) and aged (86–95 weeks of age; n = 4) Prox1 –GFP mice. Each dot is the value for one mouse. The error bars indicate mean ± s.e.m. P values were calculated by two-tailed Mann–Whitney U -tests. Anatomical positions are indicated in the bottom left or top right corner.

Article Snippet: Primary antibodies used were: anti-mouse LYVE1 (rabbit polyclonal; 11-034, Angiobio); anti-mouse VEGFR3 (goat polyclonal; AF743, R&D Systems); anti-mouse αSMA-Cy3 (mouse monoclonal, clone 1A4; C6198, Sigma); anti-mouse laminin α5 (rabbit polyclonal; EWL004, kerafast); anti-mouse CD31 (hamster monoclonal, clone 2H8; MAB1398Z, Merck); anti-mouse tyrosine hydroxylase (rabbit polyclonal; AB152, Merck); anti-mouse vesicular acetylcholine transporter (VAChT, also known as solute carrier family 18 (vesicular acetylcholine), member 3, Slc18a3, goat polyclonal; ABN100, Merck); anti-monkey LYVE1 (rabbit polyclonal; DP3500, OriGene); anti-mouse eNOS antibody (rabbit polyclonal; ab5589, Abcam); anti-mouse phospho-eNOS antibody (rabbit polyclonal; 9571, Cell Signaling); anti-mouse FOXP2 antibody (goat polyclonal; ab1307, Abcam); anti-mouse ER-TR7 antibody (rat monoclonal, clone ER-TR7; sc-73355, Santa Cruz Biotechnology); anti-mouse Col1a1 (rabbit monoclonal, clone E8F4L; 72026, Cell Signaling); and anti-mouse PDGFRα (goat polyclonal; AF1062, R&D Systems).

Techniques: Immunofluorescence, Comparison, Two Tailed Test, Staining, MANN-WHITNEY

Journal: Nature

Article Title: Increased CSF drainage by non-invasive manipulation of cervical lymphatics

doi: 10.1038/s41586-025-09052-5

Figure Lengend Snippet: a , Uniform manifold approximation and projection (UMAP) plot showing expression of Xist , a female-specific non-coding RNA, in LEC clusters from 3 male and 3 female Prox1 -GFP mice shown in Extended Data Fig. . Equal distributions of Xist positive and Xist negative cells are consistent with the sample being representative. b,c , Representative RNA in-situ hybridization images and signal counts comparing Nos3 mRNA expression in superficial cervical lymphatics scLV-1 (white dashed lines) in whole mounts from adult (8 weeks) and aged (96 weeks) Prox1 -GFP mice. Scale bars, 20 μm. In c , each dot is the value for one side of scLV-1 from one mouse. n = 6 mice (adult) and n = 5 mice (aged) from three independent experiments. Error bars indicate mean ± s.e.m. P values calculated by two-tailed Welch’s t-test. d,e , Confocal microscopic images and measurements of immunofluorescence staining for eNOS and phosphorylated-eNOS (phos-eNOS) in superficial cervical lymphatics scLV-1 in tissue whole mounts from adult (8 weeks) and aged (96 weeks) Prox1- GFP mice. Scale bars, 100 µm. Each dot is the value for one side of scLV-1 from one mouse. n = 7 (adult, eNOS), n = 6 (adult, Phos-eNOS) mice and n = 5 (aged, eNOS), n = 6 (aged, Phos-eNOS) mice from three independent experiments. Error bars indicate mean ± s.e.m. P values calculated by two-tailed Welch’s t-test. f,g , Confocal microscopic images of immunofluorescence staining ( f ) and uniform manifold approximation and projection (UMAP) plot ( g ) of FOXP2 expression in superficial cervical lymphatics scLV-1 (white dashed lines) in tissue whole mounts from adult (8 weeks) Prox1- GFP mice. Green dashed line boxes in f mark regions enlarged in the right three panels. Diverse features of FOXP2 expression in LEC are marked by colored arrowheads: red ( Foxp2 high LEC), pink ( Foxp2 low LEC), and yellow ( Foxp2 high LEC in valve). Scale bars, 50 μm. Representative of n = 3 mice from three independent experiments. FOXP2 immunofluorescence was variably strong in luminal LEC but consistently strong in valve LEC ( f ), which fits with the UMAP plot that shows two separate Foxp2 high LEC clusters ( g ). h-k , Immunofluorescence images of whole mounts showing staining for Prox1 -GFP and LYVE1 in initial lymphatics (with blunt ends, red arrowheads) and pre-collecting lymphatics (with valves, yellow arrowheads) of regional cervical lymphatics ( h ). White dashed line box marks the region enlarged in the left lower corner. Scale bar, 500 μm. Representative of n = 3 mice from three independent experiments. Immunofluorescence images of whole mounts showing Prox1 -GFP in all lymphatics but not in a facial vein; αSMA staining of smooth muscle in a collecting lymphatic and facial vein but not in a pre-collecting lymphatic; and LYVE1 staining in a pre-collecting lymphatic (white arrowheads and dashed line outline) but not in the other vessels ( i ). Scale bar, 200 μm. Representative of n = 3 mice from three independent experiments. Dot plot graph showing superficial cervical collecting lymphatics that are larger in diameter than pre-collecting lymphatics in adult Prox1 -GFP mice ( j ). Each dot is the value for one lymphangion from one side of cervical lymphatics. n = 6 mice from three independent experiments. Error bars indicate mean ± s.e.m. a.u., arbitrary unit. P values calculated by two-tailed Welch’s t-test. The immunofluorescence staining ( h,i ) fits with the strong Lyve1 mRNA expression restricted to the regional cervical (initial and pre-collecting) LEC cluster in the uniform manifold approximation and projection (UMAP) plot ( k ).

Article Snippet: Primary antibodies used were: anti-mouse LYVE1 (rabbit polyclonal; 11-034, Angiobio); anti-mouse VEGFR3 (goat polyclonal; AF743, R&D Systems); anti-mouse αSMA-Cy3 (mouse monoclonal, clone 1A4; C6198, Sigma); anti-mouse laminin α5 (rabbit polyclonal; EWL004, kerafast); anti-mouse CD31 (hamster monoclonal, clone 2H8; MAB1398Z, Merck); anti-mouse tyrosine hydroxylase (rabbit polyclonal; AB152, Merck); anti-mouse vesicular acetylcholine transporter (VAChT, also known as solute carrier family 18 (vesicular acetylcholine), member 3, Slc18a3, goat polyclonal; ABN100, Merck); anti-monkey LYVE1 (rabbit polyclonal; DP3500, OriGene); anti-mouse eNOS antibody (rabbit polyclonal; ab5589, Abcam); anti-mouse phospho-eNOS antibody (rabbit polyclonal; 9571, Cell Signaling); anti-mouse FOXP2 antibody (goat polyclonal; ab1307, Abcam); anti-mouse ER-TR7 antibody (rat monoclonal, clone ER-TR7; sc-73355, Santa Cruz Biotechnology); anti-mouse Col1a1 (rabbit monoclonal, clone E8F4L; 72026, Cell Signaling); and anti-mouse PDGFRα (goat polyclonal; AF1062, R&D Systems).

Techniques: Expressing, RNA In Situ Hybridization, Two Tailed Test, Immunofluorescence, Staining