goat anti mouse flt4  (R&D Systems)

Journal: bioRxiv

Article Title: PROX1 loss in adult mouse Schlemm’s canal causes permanent ocular hypertension

doi: 10.1101/2025.11.13.688015

Figure Lengend Snippet: Human and mouse SC endothelial cells share a lymphatic-like hybrid phenotype mediated by PROX1. a- Whole mount immunostaining and cryo cross-sections of mouse ( a ) and human ( b ) Schlemm’s canals revealed robust expression of the lymphatic markers PROX1 and FLT4. (c) PROX1 and CCL21 expression were reduced in primary human SC endothelial cells treated with PROX1 siRNA, while levels of the universally expressed endothelial gene TEK were unchanged when measured by real-time qPCR (siControl n = 6, si PROX1 n = 6). ( d , quantified in e ) Western blot revealed reduced expression of FLT4 protein in siPROX1-treated human SC cells (n = 3 per group). ( f ) Reduced expression of lymphatic genes and increased expression of blood endothelial genes were detected in si PROX1 -treated SC cells by RNA sequencing, accompanied by increased expression of TGFB-signaling genes and reduction in cell proliferation markers (n = 3 per group). ( g , quantified in h ) Confocal microscopy of eye flat mounts revealed reduced PROX1 and FLT4 expression in Prox1 flox/flox ; Cdh5 CreERT2 mice 4 weeks after tamoxifen induction at 8 weeks of age ( Prox1 ΔEC). While Prox1 deletion was generally robust, some mosaicism was observed, and a small number of PROX1-positive nuclei were observed in Prox1 ΔEC SC (white arrowheads, Prox1 ΔEC n = 3, Control n = 4). Dashed lines in g outline Schlemm’s canal. ( i ) No change in IOP ( Prox1 ΔEC n = 9, Control n = 7) or ( j ) Schlemm’s canal width ( Prox1 ΔEC n = 3, Control n = 4) was measured 4 weeks after tamoxifen induction. * p <0.05, ** p <0.01 as determined by 2-tailed unpaired Student’s t test. Error bars in c , e , h and i indicate ±SEM, while each point denotes an independent biological replicate.

Article Snippet: Primary antibodies used: Goat anti-human FLT4 (R&D Systems, Minneapolis MN, #AF349 1:100 dilution), rabbit anti human GAPDH (Cell Signalling, Danvers MA, #2118 1:10,000 dilution).

Techniques: Immunostaining, Expressing, Western Blot, RNA Sequencing, Confocal Microscopy, Control

Journal: bioRxiv

Article Title: PROX1 loss in adult mouse Schlemm’s canal causes permanent ocular hypertension

doi: 10.1101/2025.11.13.688015

Figure Lengend Snippet: ( a ) Schematic of experimental timeline used for nanocarrier induction. ( b ) 7 days after intracameral infusion of 4OHT-loaded nanocarriers, robust recombination was observed in eyes treated with nanocarriers labeled with FLT4 targeting peptide, while only sporadic recombination was observed in eyes receiving untargeted 4OHT nanocarriers. ( c ) SC-specific recombination efficiency was further increased by a second injection of FLT4-targeting nanocarriers 24h after the first. ( d ) confirming specificity of the system, no recombination was observed in retinal or choroidal capillaries of eye cryosections prepared from Rosa26 mTmG ; Cdh5 -CreERT2 mice 7 days after receiving two intracameral injections of targeted 4OHT or blank nanocarriers.

Article Snippet: Primary antibodies used: Goat anti-human FLT4 (R&D Systems, Minneapolis MN, #AF349 1:100 dilution), rabbit anti human GAPDH (Cell Signalling, Danvers MA, #2118 1:10,000 dilution).

Techniques: Labeling, Injection

Journal: bioRxiv

Article Title: PROX1 loss in adult mouse Schlemm’s canal causes permanent ocular hypertension

doi: 10.1101/2025.11.13.688015

Figure Lengend Snippet: ( a ) Experimental outline used for generation and analysis of endothelial-specific Flt4 knockout mice ( Flt4 ΔEC). ( b , quantified in c & d ) Confocal microscopy revealed loss of FLT4 immunostaining and similar PECAM1-positive SC size 5 months after Flt4 deletion. Control, n = 5, Flt4 ΔEC, n = 3. ( e ) No difference in IOP was observed in between Flt4 ΔEC mice and control littermates at 7 months of age. Control, n = 5, Flt4 ΔEC, n = 4. Statistical comparisons in c - e were performed using a 2-tailed Student’s t test. * p < 0.05. Error bars indicate ±SEM, while each point represents an independent biological replicate.

Article Snippet: Primary antibodies used: Goat anti-human FLT4 (R&D Systems, Minneapolis MN, #AF349 1:100 dilution), rabbit anti human GAPDH (Cell Signalling, Danvers MA, #2118 1:10,000 dilution).

Techniques: Knock-Out, Confocal Microscopy, Immunostaining, Control

Journal: Nature

Article Title: Dynamic cytoskeletal regulation of cell shape supports resilience of lymphatic endothelium.

doi: 10.1038/s41586-025-08724-6

Figure Lengend Snippet: Fig. 2 | Morphology and remodelling of intercellular overlaps between capillary LECs. a, Constructs for mosaic multicolour labelling of LECs using membrane-localized fluorescent proteins. b, Whole-mount immunofluorescence of mosaically labelled dermal LECs in a 6-week-old iMb2-Mosaic;Vegfr3-creERT2 mouse after 4-OHT treatment at 3 weeks, showing lobate shape in LYVE1+ capillaries and elongated shape in LYVE1− collectors (arrowheads). c, Whole-mount immunofluorescence of embryonic back skin (E17) or ear skin at indicated postnatal stages in iMb2-Mosaic;Vegfr3-creERT2 mice. d–f, Dermal LEC and vessel parameters, represented as mean ± s.d.: cell size (d, n = 24, 17 and 20 cells per respective stage), lobe number (e, n = 24, 48 and 20 cells per respective stage) and average lymphatic capillary width (f, n = 7, 5 and 9 mice per respective stage). Ordinary one-way ANOVA. g, Immunofluorescence of ear skin of a 12-week-old iMb2-Mosaic;Vegfr3-creERT2 mouse showing LYVE1 at LEC overlaps, with corresponding intensity plot. h, Double staining for cell surface and total LYVE1 (left), or with intradermally

Article Snippet: Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology and archaeology Animals and other organisms Clinical data Dual use research of concern Plants Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used The following antibodies were used for whole mount immunofluorescence (dilution 1:100-1:500): chicken anti-GFP (ab13970, Abcam), goat anti-mouse VEGFR3 (AF743, R&D Systems), goat anti-mouse VE-cadherin (R&D Systems, AF1002), goat anti-mouse PECAM1 (R&D, Systems AF3628), mouse anti-HA tag, Alexa Fluor 647 (Cell Signalling Technology, 6E2), rabbit anti-alpha tubulin (Abcam, ab52866), rabbit anti-GFP (A11122, Thermo Fisher Scientific), rabbit anti-DsRed (Takara Bio, 632496), rabbit anti-mouse LYVE1 (Reliatech, 103-PA50AG), rabbit anti-mouse CLDN5 (Invitrogen, 34-1600), rat anti-mouse PECAM1 (553370, BD Pharmingen), rat anti-mouse LYVE1-Alexa FluorTM 488, Clone ALY7 (Invitrogen, 53-0443-82), rat anti-mouse LYVE1 (R&D Systems (MAB2125), rat Anti-Mouse CD29 Clone 9EG7 (BD Pharmingen, 553715) All secondary antibodies were conjugated to Cy3(JIR, 712-165-153),(JIR, 711-166-152), Dylight 405 (JIR, 712-475-153), Alexa Fluor 488 (JIR, 703-545-155), (JIR, 712-545-153), (JIR, 711-545-152), Alexa Fluor 594 (JIR, 705-585-147), Alexa Fluor 647(JIR, 712-605-153),(JIR, 705-605-147),(JIR, 711-605-152) or Alexa Fluor 680 (JIR, 705-625-147) were raised in donkey and obtained from Jackson ImmunoResearch(JIR) .

Techniques: Construct, Membrane, Immunofluorescence, Double Staining