pan endothelial cell antigen antibody  (Novus Biologicals)

Journal: bioRxiv

Article Title: OBESITY-INDUCED ENDOTHELIAL FENESTRATION AND CAPILLARY LEAKAGE CONTRIBUTE TO INCREASED PAIN SENSATION

doi: 10.64898/2026.03.13.711502

Figure Lengend Snippet: (A) Schematic diagram of skin vasculature illustrates the organization of blood vessels in the skin layers. In the deep dermis, there are large-diameter blood vessels, including arteries and veins. The intermediate dermis contains arterioles and venules that branch from these arteries and veins, forming an intricate network. Lymphatic vessels are also located in the intermediate dermis. In the superficial dermis, capillaries form a highly branched network. (B) A representative maximum projection image from a whole-mount immunohistochemical analysis of mouse ear skin is presented. This analysis uses the pan-endothelial cell (EC) marker PECAM-1 to visualize skin vasculature from the deep to the superficial dermis. Different colors in the image indicate varying depths (Z-depth) within the dermis. Scale bar: 100 μm. (C) Representative whole-mount images of PECAM-1 + vasculature in the superficial, intermediate, and deep dermis of mouse ear skin are shown. Scale bars: 100 μm. (D) Experimental outline for generating diet-induced obesity (DIO) mice. Mice were fed either regular diet (10 Kcal % fat) or high-fat diet (60 Kcal % fat) from 6 weeks-of-age to 22 weeks-of-age. (E) Representative whole-mount images of the superficial dermal vasculature in the ear skin of control and DIO mice at 22 weeks-of-age, labeled with antibodies for the vascular smooth muscle cell marker αSMA (green or white), the vascular permeability marker PLVAP (MECA-32, red or white), along with PECAM-1 (blue or white) are presented. Scale bars: 100 μm. (F) Quantification of PLVAP + /PECAM-1 + capillaries in the superficial dermal vasculature from control and DIO mice is shown. The sample size is N = 6 in each group. (G) Representative transmission electron microscopy images of capillary ECs in the superficial dermis from control and DIO mice are presented. The dotted box regions in the left panels are magnified in the right panels. Fenestrae were observed only in DIO capillary ECs (arrowheads). Scale bars: 200 nm. (H) Quantification of endothelial fenestration in control and DIO capillary ECs is provided, showing both the number and percentage of non-fenestrated and fenestrated ECs. The sample sizes are as follows: N = 18 in control, N = 27 in DIO. Results are shown as the mean ± SEM. *p<0.05. P values were determined by the parametric two-tailed t test. The schematic diagrams and graphic summary were partially created with BioRender.com .

Article Snippet: A neutralizing anti-PLVAP antibody (clone MECA-32, BioXCell, BE0200) or a rat IgG2a isotype control (BioXCell, BE0089) was diluted to a concentration of 2.11 mg/ml in a 0.9% sodium chloride solution (Millipore Sigma, S8776).

Techniques: Immunohistochemical staining, Marker, Control, Labeling, Permeability, Transmission Assay, Electron Microscopy, Two Tailed Test

Journal: bioRxiv

Article Title: OBESITY-INDUCED ENDOTHELIAL FENESTRATION AND CAPILLARY LEAKAGE CONTRIBUTE TO INCREASED PAIN SENSATION

doi: 10.64898/2026.03.13.711502

Figure Lengend Snippet: (A) Schematic diagram illustrates capillary ECs in the superficial dermal vasculature with or without a neutralizing anti-PLVAP antibody. In DIO capillary ECs, fenestrae form, which facilitates molecular leakage from blood to tissue (left). The anti-PLVAP antibody binds to the PLVAP protein, possibly obstructing the fenestrae and inhibiting molecular leakage (right). (B) Schematic diagram illustrating the administration of the anti-PLVAP antibody into DIO mice from 20 weeks-of-age to 22 weeks-of-age using an osmotic pump. (C) Illustration shows intravital imaging of mouse ear skin, along with representative images of dextran extravasation from superficial capillaries. Lectin (green) labels capillaries, and dextran (40 kDa, red) is visible inside the capillaries immediately after injection (t=0), gradually extravasating thereafter (t=10). Scale bars: 100 μm. (D) Representative time-course images show the extravasation kinetics of dextran (40 kDa) in control skin, DIO skin treated with saline, and DIO skin treated with the neutralizing anti-PLVAP antibody. Lectin labels capillaries (green) in the superficial dermis. Time-course rainbow color images show the intensity of dextran. The amount of extravasated dextran is quantified based on the intensity of the dextran signal outside the lectin + capillaries. Scale bars: 100 μm. (E) Changes in dextran (40 kDa) extravasation are shown for control skin (blue), DIO skin treated with saline (red), and DIO skin treated with the neutralizing anti-PLVAP antibody (green). (F) Quantitative measurements of dextran (40 kDa) extravasation at the 4-minute mark are shown. The sample sizes are as follows: N = 13 in control, N = 11 in DIO + Saline, N = 9 in DIO + PLVAP ab. Results are shown as the mean ± SEM. *p<0.05, **p<0.01. P values were determined by the parametric two-tailed t test. The schematic diagrams and graphic summary were partially created with BioRender.com .

Article Snippet: A neutralizing anti-PLVAP antibody (clone MECA-32, BioXCell, BE0200) or a rat IgG2a isotype control (BioXCell, BE0089) was diluted to a concentration of 2.11 mg/ml in a 0.9% sodium chloride solution (Millipore Sigma, S8776).

Techniques: Imaging, Injection, Control, Saline, Two Tailed Test

Journal: bioRxiv

Article Title: OBESITY-INDUCED ENDOTHELIAL FENESTRATION AND CAPILLARY LEAKAGE CONTRIBUTE TO INCREASED PAIN SENSATION

doi: 10.64898/2026.03.13.711502

Figure Lengend Snippet: (A) Schematic diagram illustrates the changes in vascular structure and sensory functions in the skin between control and DIO mice. In the skin of DIO mice, capillary ECs become fenestrated, leading to increased vascular permeability. Additionally, DIO mice exhibit enhanced pain behavior and sensory hypersensitivity . (B) Illustration shows the implantation of an osmotic pump in sensory neuron-specific Pirt-GCaMP3 calcium reporter mice. This pump is used to administer saline, the IgG control, or the neutralizing anti-PLVAP antibody. The sample sizes are as follows: N = 6 in Pirt-GCaMP3 mice on a control diet (control), N = 8 in Pirt-GCaMP3 mice with DIO receiving saline (DIO + Saline), N = 5 in Pirt-GCaMP3 mice with DIO receiving IgG control (DIO + IgG control), N = 8 in Pirt-GCaMP3 mice with DIO receiving the anti-PLVAP antibody (DIO + PLVAP Ab). (C) Illustrations depict the capsaicin-mediated acute pain behavior assay (left) and ex vivo Ca 2+ imaging of peripheral terminals of nociceptive neurons located in the epidermis of the ear skin (right). (D) Total forelimb wiping responses following capsaicin application are shown for control, DIO + Saline, DIO + IgG control, and DIO + PLVAP Ab. (E) Quantification of Ca 2+ responses within the ear skin of control mice, DIO mice with saline, DIO mice with the IgG, and DIO mice with the neutralizing anti-PLVAP antibody is shown. The Ca 2+ transients were normalized by the baseline Ca 2+ transient (ΔF/F 0 ). (F) The integrated Ca 2+ transient (ΔF/F0) was calculated as the area under the curve (AUC). Results are shown as the mean ± SEM. *p<0.05, ***p<0.001. P values were determined by the parametric two-tailed t test. The schematic diagrams and graphic summary were partially created with BioRender.com .

Article Snippet: A neutralizing anti-PLVAP antibody (clone MECA-32, BioXCell, BE0200) or a rat IgG2a isotype control (BioXCell, BE0089) was diluted to a concentration of 2.11 mg/ml in a 0.9% sodium chloride solution (Millipore Sigma, S8776).

Techniques: Control, Permeability, Saline, Behavioral Assay, Ex Vivo, Imaging, Two Tailed Test

Journal: bioRxiv

Article Title: OBESITY-INDUCED ENDOTHELIAL FENESTRATION AND CAPILLARY LEAKAGE CONTRIBUTE TO INCREASED PAIN SENSATION

doi: 10.64898/2026.03.13.711502

Figure Lengend Snippet: (A) Representative section immunohistochemical images of ear skin from control mice, DIO mice treated with saline, and DIO mice treated with the neutralizing anti-PLVAP antibody are presented. This assay uses the antibodies for FOXO1 (green), the keratinocyte marker K14 (red), along with the nuclear marker TOPRO3 (blue). Each inset displays the pattern of FOXO1 expression in a single keratinocyte. Dashed lines indicate the boundary between the epidermis and the dermis. “Epi” indicates the epidermis; “D” indicates the dermis. Scale bars: 20 μm. (B) Quantification of nuclear FOXO1 expression in keratinocytes is provided. The percentages of nuclear FOXO1 expression within the total FOXO1 expression in keratinocytes are presented. The sample sizes are as follows: N = 5 in control, N = 5 in DIO + Saline, N = 7 in DIO + PLVAP Ab. (C) Representative X-gal staining images of ear skin from NGF-LacZ control mice, DIO mice with saline, and DIO mice with the neutralizing anti-PLVAP antibody (blue) are presented. Dashed lines indicate the boundary between the epidermis and the dermis. Scale bars: 50 μm. (D) Quantification of the LacZ-positive area in the epidermis is provided. The sample sizes are as follows: N = 9 in control, N = 15 in DIO + Saline, N = 9 in DIO + PLVAP Ab. (E) Graphical summary illustrates how vascular hyperpermeability leads to sensory hypersensitivity in DIO skin. Increased permeability in the superficial dermal capillaries facilitates the diffusion of insulin into the epidermis, activating insulin signaling in epidermal keratinocytes. This activation leads to NGF upregulation in these keratinocytes, which in turn promotes sensory hypersensitivity in DIO skin. A neutralizing anti-PLVAP antibody reduces the diffusion of insulin, thereby decreasing NGF expression in the epidermal keratinocytes and alleviating sensory hypersensitivity. Results are shown as the mean ± SEM. *p<0.05, **p<0.01. P values were determined by the parametric two-tailed t test. The schematic diagrams and graphic summary were partially created with BioRender.com .

Article Snippet: A neutralizing anti-PLVAP antibody (clone MECA-32, BioXCell, BE0200) or a rat IgG2a isotype control (BioXCell, BE0089) was diluted to a concentration of 2.11 mg/ml in a 0.9% sodium chloride solution (Millipore Sigma, S8776).

Techniques: Immunohistochemical staining, Control, Saline, Marker, Expressing, Staining, Permeability, Diffusion-based Assay, Activation Assay, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Annexin A2 interferes with complement regulation within the glomerulus

doi: 10.1016/j.jbc.2025.110657

Figure Lengend Snippet: Interactions between annexin A2 and the different glomerular cell types. A , expression of annexin A2 on mesangial cells, glomerular endothelial cells (GEnC), and podocytes was examined by flow cytometry. Annexin A2 was detected on all three cell types. B , immunofluorescence microscopy for annexin A2 in GEnCs indicated that the protein is also expressed within the cells. C , When injured/apoptotic and healthy cells were distinguished by side scatter and forward scatter, more annexin A2 was seen on the surface of the apoptotic mesangial cells and podocytes, whereas expression was comparable on the injured and healthy GEnC populations. D , when rA2 was added to glomerular cells exposed to 10% normal mouse serum, a trend towards more C3b was deposited on the surface of GEnCs compared to cells exposed to serum without rA2, and greater C3 was deposited on podocytes. Groups were compared using an unpaired Student t test with a two-tailed p value.

Article Snippet: Antibodies used in the experiments included a rabbit polyclonal antibody to human annexin A2 (Proteintech; #11256-1-AP, 1:250 dilution), IgG1 monoclonal anti-AnxA2 clone Z014 (Invitrogen; #034400, 2 μg per sample), IgG1 isotype control clone MG1-45 (Abcam; # ab18448, 2 μg per sample), a Cy3-conjugated polyclonal goat anti-mouse IgG (Jackson Immuno; 1:100 dilution), an Alexa Fluor Plus 647 labeled goat anti-mouse IgG (Invitrogen; #A32728, 1:1000 dilution), a FITC-labeled goat polyclonal antibody to mouse C3 (MP Biomedicals; #55500; 10 μg/ml for tissue staining), Digoxin (DIG) labeled anti-mouse C9 (generously provided by Cees van Kooten, Leiden University Medical Center; 2 μg/ml for tissue staining), DyLight 405 IgG fraction monoclonal mouse anti-Digoxin (Jackson Immuno; #200-472-156, 2.7 μg/ml for tissue staining), and MECA32, a pan-endothelial cell antigen antibody (Novus Biologicals; NB100-77668, 0.25 μg/100 μl reaction containing 10 6 cells).

Techniques: Expressing, Flow Cytometry, Immunofluorescence, Microscopy, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Annexin A2 interferes with complement regulation within the glomerulus

doi: 10.1016/j.jbc.2025.110657

Figure Lengend Snippet: Recombinant annexin A2 increases glomerular C3b deposition in vivo . A , fH +/− mice were injected with 75 μg recombinant annexin A2. After 24 h, the kidneys were harvested and examined by immunofluorescence microscopy. Original magnification ×600. B , deposition of C3b and iC3b/C3d was quantified, and the abundance of both activation fragments was increased compared to mice injected with phosphate-buffered saline (PBS). Injection of the mice with rA2 did not increase C9 deposits in the glomeruli. C , kidneys of rA2-injected and control mice were digested with collagenase. Endothelial cells were identified by surface expression of MECA32, and C3b deposits on the cells were measured by flow cytometry. D , endothelial cells from rA2-injected mice had significantly more C3b deposited on their surface compared to PBS-injected mice. Groups were compared using an unpaired Student t test with a two-tailed p value.

Article Snippet: Antibodies used in the experiments included a rabbit polyclonal antibody to human annexin A2 (Proteintech; #11256-1-AP, 1:250 dilution), IgG1 monoclonal anti-AnxA2 clone Z014 (Invitrogen; #034400, 2 μg per sample), IgG1 isotype control clone MG1-45 (Abcam; # ab18448, 2 μg per sample), a Cy3-conjugated polyclonal goat anti-mouse IgG (Jackson Immuno; 1:100 dilution), an Alexa Fluor Plus 647 labeled goat anti-mouse IgG (Invitrogen; #A32728, 1:1000 dilution), a FITC-labeled goat polyclonal antibody to mouse C3 (MP Biomedicals; #55500; 10 μg/ml for tissue staining), Digoxin (DIG) labeled anti-mouse C9 (generously provided by Cees van Kooten, Leiden University Medical Center; 2 μg/ml for tissue staining), DyLight 405 IgG fraction monoclonal mouse anti-Digoxin (Jackson Immuno; #200-472-156, 2.7 μg/ml for tissue staining), and MECA32, a pan-endothelial cell antigen antibody (Novus Biologicals; NB100-77668, 0.25 μg/100 μl reaction containing 10 6 cells).

Techniques: Recombinant, In Vivo, Injection, Immunofluorescence, Microscopy, Activation Assay, Saline, Control, Expressing, Flow Cytometry, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Annexin A2 interferes with complement regulation within the glomerulus

doi: 10.1016/j.jbc.2025.110657

Figure Lengend Snippet: Mice deficient in annexin A2 are protected from cyclosporine-induced kidney injury. A , mice with targeted deletion of the gene for annexin A2 ( A2 −/− mice) and wild-type control mice were injected subcutaneously with cyclosporine (CsA) for 2 weeks. A , serum urea nitrogen (SUN) was measured as a marker of kidney function. SUN levels were significantly increased in wild-type mice injected with CsA, but did not increase in the A2 −/− mice. Glomerular deposition of C3b ( B ) and iC3b/C3d ( C ) were quantified by fluorescence microscopy, and the abundance of both activation fragments were similar in the wild-type and A2 −/− mice. D , representative images stained for C3b ( green ) and iC3b/C3d ( red ) are shown. Original magnification ×200. Glomeruli are indicated with arrowheads . E , endothelial extracellular vesicles (EVs) were isolated from the plasma of CsA treated mice, and C3b deposits on the surface of the EVs were measured by flow cytometry. The abundance of C3b on the EVs from wild-type mice were increased in mice treated with CsA, whereas it did not increase in A2 −/− mice treated with CsA. F , the number of endothelial EVs per μl of plasma was significantly higher in the A2 −/− mice treated with CsA compared to the other groups ( p < 0.01 for A2 −/− CsA versus the other groups). Groups were compared using an ANOVA, with a Tukey’s multiple comparison test.

Article Snippet: Antibodies used in the experiments included a rabbit polyclonal antibody to human annexin A2 (Proteintech; #11256-1-AP, 1:250 dilution), IgG1 monoclonal anti-AnxA2 clone Z014 (Invitrogen; #034400, 2 μg per sample), IgG1 isotype control clone MG1-45 (Abcam; # ab18448, 2 μg per sample), a Cy3-conjugated polyclonal goat anti-mouse IgG (Jackson Immuno; 1:100 dilution), an Alexa Fluor Plus 647 labeled goat anti-mouse IgG (Invitrogen; #A32728, 1:1000 dilution), a FITC-labeled goat polyclonal antibody to mouse C3 (MP Biomedicals; #55500; 10 μg/ml for tissue staining), Digoxin (DIG) labeled anti-mouse C9 (generously provided by Cees van Kooten, Leiden University Medical Center; 2 μg/ml for tissue staining), DyLight 405 IgG fraction monoclonal mouse anti-Digoxin (Jackson Immuno; #200-472-156, 2.7 μg/ml for tissue staining), and MECA32, a pan-endothelial cell antigen antibody (Novus Biologicals; NB100-77668, 0.25 μg/100 μl reaction containing 10 6 cells).

Techniques: Control, Injection, Marker, Fluorescence, Microscopy, Activation Assay, Staining, Isolation, Clinical Proteomics, Flow Cytometry, Comparison