Frozen cross-sections were used in these experiments. Specimens were blocked with 5% BSA for 30 minutes and incubated with a primary antibody for 2 hours at 37°C. Hindlimb nerves were identified by using an anti-mouse S-100 antibody (1:100, Abcam). Vessel smooth muscles were identified using an anti-rabbit α-SM-actin (1:100, Abcam). Capillary ECs and collateral ECs were identified using an anti-mouse CD31 antibody (1:100, Abcam). The appropriate Alexa Fluor® 647 and Alexa Fluor® 488 secondary antibodies (1:500, CST) were used. Nuclei were stained using DAPI. All sections were photographed under a microscope (Olympus).
Anti mouse cd31 antibody
Manufactured by Abcam
Sourced in United States, United Kingdom
The Anti-mouse CD31 antibody is a primary antibody that binds to the CD31 (also known as PECAM-1) protein, which is expressed on the surface of endothelial cells, platelets, and some leukocytes. This antibody can be used in various applications to detect and study the CD31 protein in mouse biological samples.
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Lab products found in correlation
Anti mouse α sma antibody, by Abcam (1 mentions)
Anti human αsma antibody, by Agilent Technologies (1 mentions)
Texas red conjugated secondary antibody, by Vector Laboratories (1 mentions)
Oct compound, by Sakura Finetek (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
Avidin biotin blocking kit, by Vector Laboratories (1 mentions)
Biotinylated anti mouse secondary antibody, by Agilent Technologies (1 mentions)
Envision detection system, by Agilent Technologies (1 mentions)
Goat anti mouse igg fitc secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Anti cd68 antibody, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Antifade mounting medium, by Beyotime (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Enhanced chemoluminescence system, by Clinx (1 mentions)
Rabbit anti phosphorylated erk, by Cell Signaling Technology (1 mentions)
Rabbit anti erk, by Cell Signaling Technology (1 mentions)
Rabbit anti akt, by Cell Signaling Technology (1 mentions)
Rabbit anti phosphorylated akt ser 473, by Cell Signaling Technology (1 mentions)
Rabbit anti α smooth muscle actin antibody, by Abcam (1 mentions)
Mouse anti β catenin antibody, by Merck Group (1 mentions)
11 protocols using anti mouse cd31 antibody
1
Immunofluorescent Nerve and Vascular Mapping
2
Quantifying Liver Cell Markers
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The slices were permeated with 0.5% Triton X-100. Primary antibodies against rat αvβ3 integrin (diluted 1:50, Abcam, United Kingdom), anti-mouse α-SMA antibody (diluted 1:400, Abcam, United Kingdom), and anti-mouse CD31 antibody (diluted 1:50, Abcam, United Kingdom) were used. Secondary antibodies included Alexa Fluor 647-conjugated goat anti-rabbit (diluted 1:400, Abcam, United Kingdom) and FITC-conjugated mouse anti-rat secondary antibodies (diluted 1:400, Abcam, United Kingdom). The liver sections were incubated with mixed primary antibody, mixed secondary antibody, and DAPI. After washing with PBS, the slides were mounted using an anti-fluorescence quencher. Multicoloured fluorescent staining images were analysed via CLSM. The mean fluorescence densities of liver sections were calculated using Image-Pro Plus 6.0. For each group, three amplifying fields (400) were randomly chosen to conduct a semi-quantitative analysis of integrin αvβ3, α-SMA, and CD31 expression levels.
3
Immunofluorescence Staining of Vascular Markers
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Wounded tissues were embedded in optical cutting compound (OCT compound, Sakura Finetek, Tokyo, Japan) and cut into 4‐μm‐thick sections. To eliminate OCT compound, sections were washed thrice with PBS and incubated in 20% normal goat serum to block non‐specific binding. Sections were probed with anti‐mouse CD31 antibody (Abcam) for 2 h at RT, followed by FITC‐conjugated secondary antibody for 1 h. After two washes, anti‐human α‐SMA antibody (Dako, DK) was added, followed by Texas Red‐conjugated secondary antibody (Vector Laboratories). Thereafter, samples were mounted with Vectashield mounting medium (Vector Laboratories with DAPI) and observed using a Nuance Multiplex Biomarker Imaging System (Cambridge Research Instrumentation).
4
Immunohistochemical Analysis of Tumor Vasculature and Immune Cells
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The tumor tissues were fixed with 4% paraformaldehyde, and processed for immunohistochemical examination of tumor blood vessels, apoptosis (TUNEL) and immune cells. To prevent nonspecific binding of antibodies, tumor slides were blocked with the protein blocking buffer containing 4% FBS, 1% normal goat serum, and 0.01% Tween-20 in PBS for 1 h at 4 °C. For TUNEL analysis, the procedure was according to Colorimetric TUNEL Apoptosis Assay (Beyotime, China). For other detection, the slides were subsequently incubated with anti-mouse CD31 antibody (Abcam) or FITC-conjugated anti-mouse F4/80 and PE-conjugated anti-mouse CD86 or PE-conjugated anti-mouse CD206 antibodies (eBioscience, CA, USA), PE-conjugated SIINFEKL/H-2Kb peptide-MHC tetramers (Creative peptides, USA) at 4 °C. After being washed thoroughly with PBS, the slides were stained with Hoechst 33342 to identify the loci of cell nuclei. Images were captured with the microscope and CLSM. The percentage of CD31 positive area was obtained from the ratio of the CD31 positive area to the total cell area. The above experiments are provided by Servicebio Biotechnology Co., Ltd. (Wuhan, China).
5
Quantifying Angiogenesis via Immunohistochemistry
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Immunohistochemical staining for IGFBP-5 was carried out on 4-μm formalin-fixed, paraffin-embedded tissue sections. After antigen retrieval in 10 mM sodium citrate buffer (pH 6.5) in a microwave followed by blocking with an Avidin/biotin blocking kit (Vector Labs, Burlingame, CA, USA), tissue slides were stained with anti-IGFBP-5 antibody (Santa Cruz Biotechnologies, Dallas, TX, USA) and biotinylated anti-mouse secondary antibody (DAKO, Denmark). IGFBP-5 was detected with avidin-conjugated HRP (DAKO) and images were scanned with a Scan Scope (Aperio, Heidelberger, Germany). To quantify angiogenesis, microvessel density was determined by counting CD31-positive vessels as described previously46 (link). In brief, 8-μm thick sections of fresh-frozen tumor samples were fixed and incubated with anti-mouse CD31 antibody (Abcam, Cambridge, UK) at 4 °C overnight. CD31 staining was detected with an Envision detection system (DAKO).
6
Quantifying Vascular Adhesion Molecules by Immunofluorescence
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OCT (Sakura Finetek, Tokyo, Japan)‐embedded tissue cryosections (9‐µm thick) were fixed at −20°C in methanol/acetone (3:1), blocked using 1% bovine serum and stained with primary antibodies anti‐mouse ICAM‐1, ICAM‐2, vascular cell adhesion molecule‐1 (VCAM)‐1 antibodies (BD Biosciences, San Jose, CA) and anti‐mouse CD31 antibody (Abcam, Cambridge, United Kingdom). Images of at least five consecutive fields (unit area of each field, .34 mm2) were captured by observers blinded to sample identity. Identical exposure times and image settings were used within each experiment. Images were analysed with ImageJ software (NIH, Bethesda, MD) for the determination of the relative fluorescence staining intensity; regions of interest were defined based on CD31 fluorescence, and each pixel in identified regions was assigned a fluorescence intensity value (based on a scale from 0 to 255).
7
Wound Healing Tissue Collection and Analysis
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To avoid skin contraction, complete wound specimens and the normal skin tissue within 2–3 mm around the wounds were collected on days 3 and 21 post-operation. The skin tissues were fixed overnight by 4% polyoxymethylene and then dehydrated by 20% sucrose. After embedding in optimal cutting temperature compound and freezing in liquid nitrogen, the specimens were cut into 5-μm sections for immunostaining. After blocking with 5% BSA, skin sections were incubated with a mouse anti-CD31 antibody (Abcam, Cambridge, MA, USA) or anti-CD68 antibody (Abcam), followed by a goat anti-mouse IgG-FITC secondary antibody (Santa Cruz Biotechnology, Texas, USA). The nuclei were stained with DAPI (Sigma-Aldrich, St. Louis, MO USA). Images were analyzed using fluorescent microscopy.
8
Immunofluorescence Analysis of Rat Brain Tissue
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Rats were anesthetized at 1, 3, 7, and 14 days after I/R, and the brains were removed after perfusion with NS and 4% paraformaldehyde. After gradient elution with sucrose, the brains were quickly frozen and cut into 8 μm coronal thick sections. The sections were permeabilized with 0.5% Triton X-100 for 5 min, were blocked with 10% donkey serum for 1 h, and then incubated with mouse anti-CD31 antibody (1:100, Abcam, United States) overnight at 4°C. Thereafter, sections were briefly washed with PBST and incubated with donkey anti-mouse secondary antibody (1:100, Cell Signal, United States) for 1 h at 37°C. After counterstaining with 4,6-diamidino-2-phenylindole (DAPI; Beyotime, Shanghai, China) and coversliping with anti-fade mounting medium (Beyotime, Shanghai, China), the sections were observed and photographed under fluorescent microscopy and then analyzed with Image-Pro Plus 6.0 software.
9
Protein Expression and Signaling Pathways Analysis
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Cell lysates were prepared with RIPA buffer (Beyotime). Protein concentration was determined using a BCA Protein Assay kit (Pierce). Samples were separated by SDS‐PAGE, blotted onto polyvinylidene fluoride membranes, and probed with primary antibodies, followed by horseradish peroxidase–conjugated goat antirabbit IgG or goat antimouse IgG (Boster Bio Tec). β‐actin was used as a loading control. The primary antibodies included rabbit antihuman EVL (1:50; Santa Cruz Biotechnology), mouse antihuman endoglin (1:800; BD Biosciences), rabbit anti–phosphorylated Smad1/5 (1:1000; Cell Signaling), rabbit anti–phosphorylated Smad2/3 (1:500; Santa Cruz Biotechnology), rabbit anti–phosphorylated Akt (Ser 473, 1:800; Cell Signaling), rabbit anti‐Akt (1:800; Cell Signaling), rabbit anti–phosphorylated ERK (1:1000; Cell Signaling), rabbit anti‐ERK (1:1000; Cell Signaling), mouse anti‐β‐actin (1:1000; Sigma‐Aldrich), mouse anti‐CD31 antibody (1:1000; Abcam), rabbit anti–α‐smooth muscle actin antibody (1:200; Abcam), mouse anti–β‐catenin antibody (1:1000; Millipore), rabbit anti‐vimentin antibody (1:1000; Abcam). Membranes were developed using an enhanced chemoluminescence system (Clinx Science Instruments).
10
Electrospun Microfibrous Membranes for HCEC Adhesion
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