anti cd31 primary antibody  (Servicebio Inc)

Journal: Science Advances

Article Title: Glycerol-mediated nose-to-brain codelivery of anti–IL-17 and anti-CD73 antibodies enhances immunotherapy for melanoma brain metastases

doi: 10.1126/sciadv.adx7966

Figure Lengend Snippet: ( A and B ) Mice underwent brain fluorescence imaging at 0, 0.5, 1, 2, 4, 8, 12, 24, and 48 hours postintranasal delivery of Cy7-labeled a–IL-17 (Cy7–a–IL-17) and Cy5-labeled a-CD73 (Cy5–a-CD73). ( C and D ) Quantitative analysis of the fluorescence intensity for Cy7–a–IL-17 (C) and Cy5-a-CD73 (D) ( n = 3). ( E and F ) Quantitative analysis of the fluorescence intensity for Cy7–a–IL-17 (E) and Cy5–a-CD73 (F) in isolated brains at 2 hours ( n = 3). ( G ) Representative fluorescence microscopy images of brain sections 2 hours after intranasal delivery or intravenous injection of FITC–a–IL-17 (green) or Cy5–a-CD73 (red). Nuclei were counterstained with DAPI (blue). Scale bars, 20 μm. ( H to K ) Quantitative analysis of fluorescence intensity in the olfactory bulb (H), cortex (I), hippocampus (J), and cerebellum (K) ( n = 3). ( L ) Representative images of brain immunofluorescence were captured 2 hours after intranasal delivery or intravenous injection of FITC-IgG (green) to observe the distribution of antibodies in brain tissue sections. Blood vessels were stained with CD31 (red). Scale bars, 20 μm. All statistics are expressed as means ± SD. Statistical significance was assessed using one-way analysis of variance (ANOVA) with Fisher’s LSD test, where * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. ns, nonsignificant.

Article Snippet: For precise cellular localization, the sections were subjected to immunofluorescence staining: The vascular endothelium was labeled with an anti-CD31 primary antibody (catalog no. GB12063-100, Servicebio, Hubei, China), visualized with a Cy3-conjugated secondary antibody, and counterstained with DAPI for nuclei.

Techniques: Fluorescence, Imaging, Labeling, Isolation, Microscopy, Injection, Olfactory, Immunofluorescence, Staining

Journal: The Journal of General Physiology

Article Title: Beat-locked ATP microdomains in the sinoatrial node map a Ca 2+ -timed energetic hierarchy and regional pacemaker roles

doi: 10.1085/jgp.202513874

Figure Lengend Snippet: Superior SA node myocytes exhibit elevated diastolic ATP and metabolic flux compared with the inferior region. (A) 3D segmented maximum-intensity projection of a whole-mount SA node immunolabeled for CD31 (vasculature, red) and cyto-iATP (myocytes, green). The dashed line denotes the boundary between superior and inferior regions. (B) Image-processing workflow illustrating merged maximum-intensity projections, binary segmentation masks, and extraction of grayscale cyto-iATP signals used for quantitative analysis. (C) Mean cyto-iATP fluorescence intensity per myocyte, grouped by region ( N = 5 mice per region), reporting expression levels of the EGFP-tagged cyto-iATP sensor. (D) Live confocal imaging of cyto-iATP signals showing representative line-scan images and corresponding normalized fluorescence traces (F/F 0 ) from superior and inferior regions. (E and F) Summary quantification of cyto-iATP signal mass rate (E) and estimated diastolic [ATP] i (F). P values are shown above comparisons. Large circles denote per-animal means; small circles indicate individual biological replicates. N represents the number of independent mice.

Article Snippet: For immunolabeling, SA nodes were incubated for 48 h at 4°C with a goat anti-mouse CD31 primary antibody (1:50, AF3628; R&D Systems).

Techniques: Immunolabeling, Extraction, Fluorescence, Expressing, Imaging

Journal: Journal of Advanced Research

Article Title: OTUD1 delays wound healing by regulating endothelial function and angiogenesis in diabetic mice

doi: 10.1016/j.jare.2025.04.038

Figure Lengend Snippet: Endothelial OTUD1 is significantly upregulated in diabetic wound tissues. (A) Real-time qPCR analysis for mRNA levels of OTU subfamily members in the skin wound tissues from control and T2DM mice (n = 7). (B) Immunoblotting and densitometric quantification analyses illustrating OTUD1 protein expression in skin wound tissues from both control and T2DM mice (n = 3). (C) Representative immunohistochemical images and (D) quantitative analysis of showing OTUD1-positive cells (brown) in the skin wound tissues of mice. Black arrowheads indicate positive OTUD1 signals (Scale bar = 50 μm; n = 7). (E) Immunoblot and quantitative analysis of OTUD1 protein expression in HUVECs, HaCaT, HDFa, and MPM cells (n = 4). (F) Representative immunofluorescence images displaying the colocalization of CD31 (red) and OTUD1 (green) in mouse skin wound tissues, with white arrowheads showing OTUD1 and CD31 colocalization sites. Tissues were counterstained with DAPI (blue; Scale bar = 50 μm). (G) Immunofluorescence staining and (H) quantitative analysis of OTUD1-positive cells (red) in both control and HG + PA-treated HUVECs for 4 h, counterstained with DAPI (blue; Scale bar = 50 μm). (I) Time-course study of OTUD1 expression in response to HG + PA in HUVECs, including immunoblot analysis and quantitative measurement (n = 3). Data are shown as mean ± SEM. Statistical analyses were performed using a two-tailed unpaired Student's t -test (A, B, D), Welch’s t test (H), and one-way ANOVA analysis followed by Bonferroni post-hoc test (E, I). HG + PA indicates treatment with 50 mM HG and 300 μM PA, unless specified otherwise. Abbreviations: Ctrl, control; T2DM, type 2 diabetes mellitus; OTU, ovarian tumor protease; HUVECs, human umbilical vein endothelial cells; HaCaT, human keratinocytes; HDFa, human dermal fibroblasts-adult; MPMs, mouse primary peritoneal macrophages; DAPI, 4ʹ,6-diamidino-2-phenylindole; HG + PA, high glucose plus palmitic acid; and OTUD1, ovarian tumor deubiquitinase 1.

Article Snippet: After one hour of blocking with rabbit serum (G1209, Servicebio), primary antibodies against CD31 (AF3628, R&D Systems, Minnesota, USA) were incubated for the whole night at 4 °C.

Techniques: Control, Western Blot, Expressing, Immunohistochemical staining, Immunofluorescence, Staining, Two Tailed Test

Journal: Journal of Advanced Research

Article Title: OTUD1 delays wound healing by regulating endothelial function and angiogenesis in diabetic mice

doi: 10.1016/j.jare.2025.04.038

Figure Lengend Snippet: OTUD1 deficiency rescues impaired wound healing by enhancing angiogenesis and fibrosis in T2DM mice. (A) Schematic diagram illustrating the animal experiment procedure. The mice (B) FBG levels and (C) body weight were recorded from weeks 9 to 16 (n = 7). * P < 0.05 vs WT-Sham; ** P < 0.01 vs WT-Sham; *** P < 0.001 vs WT-Sham; ns, no significance. ns (green) indicates that there is no statistical significance between OTUD1 −/− -T2DM and WT-T2DM. (D) Representative wound images and (E) wound closure rates are shown (n = 5). *** P < 0.001 vs WT-Sham; ## P < 0.01 vs WT-T2DM; ns, no significance. (F) H&E staining demonstrated regenerated skin at day 12 across different groups. Scale bar = 50 μm. (G) Quantitative assessments of epidermis thickness in mice (n = 7). (H) Masson's trichrome staining and (I) quantitative analysis of collagen deposition in skin wound tissues at day 12 (Scale bar = 50 μm; n = 7). (J) Representative images and (K) quantification of CD31-positive (brown) neovascularization via immunohistochemical staining at days 3, 7, and 12 (Scale bar = 50 μm; n = 7). Black arrows indicate skin neovascularization. (L-O) Immunoblotting and quantification of OTUD1, VEGFR2, p-eNOS, and eNOS in wound tissue lysates from Sham or T2DM mice with WT or OTUD1 knockout, normalized to GAPDH (n = 4). Data are displayed as mean ± SEM. Statistical analyses were performed using two-way ANOVA analysis followed by Bonferroni post-hoc test (B, C, E) and one-way ANOVA analysis followed by Bonferroni post-hoc test (G, I, K, M-O). Abbreviations: WT, wild-type; OTUD −/− , OTUD1-knockout; HFD, high-fat diet; STZ, streptozotocin; FBG, fasting blood glucose; eNOS, endothelial nitric oxide synthase.

Article Snippet: After one hour of blocking with rabbit serum (G1209, Servicebio), primary antibodies against CD31 (AF3628, R&D Systems, Minnesota, USA) were incubated for the whole night at 4 °C.

Techniques: Staining, Immunohistochemical staining, Western Blot, Knock-Out

Journal: Journal of Advanced Research

Article Title: OTUD1 delays wound healing by regulating endothelial function and angiogenesis in diabetic mice

doi: 10.1016/j.jare.2025.04.038

Figure Lengend Snippet: Pharmacological inhibition of β-catenin reverses OTUD1 deletion-mediated recovery of delayed wound healing in db/db mice. (A) Schematic of the protocol for establishing the OTUD1 deletion mouse model in db/m or db/db mice. The mice (B) FBG levels and (C) body weight of the indicated mice at weeks 10, 12, 14, 16, 18, 20, 22, and 24 (n = 7). *** P < 0.001 vs db/m -AAV-shNC. ns (green) indicates that there is no statistical significance between db/db -AAV-shNC and db/db -AAV-shOTUD1. ns (purple) indicates that there is no statistical significance between db/db -AAV-shOTUD1 and db/db -AAV-shOTUD1-MSAB. (D) Skin wound healing images and (E) statistical analysis of wound closure rate (n = 5). ** P < 0.01 vs db/m -AAV-shNC; *** P < 0.001 vs db/m -AAV-shNC; ## P < 0.01 vs db/db -AAV-shNC; ### P < 0.001 vs db/db -AAV-shNC; && P < 0.01 vs db/db -AAV-shOTUD1; ns, no significance. (F) H&E staining images of skin wound tissues. Scale bar = 50 μm. (G) Representative Masson’s trichrome staining in skin wound tissues. Scale bar = 50 μm. (H) CD31 immunohistochemical staining of neovascularization in skin wound tissues on days 3, 7, and 12 (Scale bar = 50 μm). The black arrows denote skin neovascularization. (I) Representative immunoblotting and (J-M) quantitative analysis of OTUD1, VEGFR2, Nuc-β-catenin, p-eNOS, and eNOS protein levels in skin wound tissues from different groups (n = 4). Data are shown as mean ± SEM. Statistical analyses were performed using two-way ANOVA analysis followed by Bonferroni post-hoc test (B, C, E) and one-way ANOVA analysis followed by Bonferroni post-hoc test (J-M). Abbreviations: MSAB, ethionine sulfoxide β-methyl ester; AAV2/BI30-shOTUD1, adeno-associated virus serotype 2/BI30 carrying shOTUD1 under the CMV promoter.

Article Snippet: After one hour of blocking with rabbit serum (G1209, Servicebio), primary antibodies against CD31 (AF3628, R&D Systems, Minnesota, USA) were incubated for the whole night at 4 °C.

Techniques: Inhibition, Staining, Immunohistochemical staining, Western Blot, Virus