Electroacupuncture Treatment Device (SDZ-II, Huatuo Brand) was from Suzhou Medical Appliance Factory, China. The size of the needles was 0.35 mm (Diameter) × 40 mm (Length). Antibodies used for immunostaining in this study were purchased from Abcam (MA, USA) including anti-CD34 (Mouse monoclonal, catalog no. ab8536), anti-VEGFA (Mouse monoclonal, catalog no. ab1316), anti-VEGF receptor-2 (Rabbit polyclonal, catalog no. ab2349), anti-angiopoietin-1(Rabbit polyclonal, catalog no. ab102015), anti-DLL4 (Rabbit polyclonal, catalog no. ab217860), anti-MMP-2 (Mouse monoclonal, catalog no. ab86607), anti-TIMP-2 (Mouse monoclonal, catalog no. ab1828), anti-integrin-β (Mouse monoclonal, catalog no. ab24693), anti-p75 NGF receptor (Rabbit monoclonal, catalog no. ab52987), anti-Sema3A (Rabbit polyclonal, catalog no. ab23393) and anti-Neuropilin 1 (Rabbit monoclonal, catalog no. ab81321). Mouse TNF-alpha ELISA kit was obtained from eBioscience (Thermo Fisher, MA, USA).
Anti dll4
Manufactured by Abcam
Sourced in United States
Anti-DLL4 is a laboratory reagent used in research applications. It is an antibody that binds to the Delta-like protein 4 (DLL4), which is a critical component of the Notch signaling pathway. This reagent can be utilized in various experimental techniques to study the role of DLL4 in cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Anti vegfa, by Abcam (2 mentions)
Anti notch1, by Abcam (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Anti timp2, by Abcam (1 mentions)
Anti cd34, by Abcam (1 mentions)
Tnf alpha mouse elisa kit, by Thermo Fisher Scientific (1 mentions)
Anti vegf receptor 2, by Abcam (1 mentions)
Anti angiopoietin 1, by Abcam (1 mentions)
Anti integrin β, by Abcam (1 mentions)
Anti p75 ngf receptor, by Abcam (1 mentions)
Anti sema3a, by Abcam (1 mentions)
Anti mmp2, by Abcam (1 mentions)
Alexa fluor 488 or 594 secondary antibody, by Thermo Fisher Scientific (1 mentions)
Mouse monoclonal anti mucin5ac, by Santa Cruz Biotechnology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Fv1000, by Olympus (1 mentions)
Quick start bradford protein assay kit, by Bio-Rad (1 mentions)
Immobilon, by Merck Group (1 mentions)
Ripa buffer, by Santa Cruz Biotechnology (1 mentions)
Ez capture, by ATTO Corporation (1 mentions)
8 protocols using anti dll4
1
Electroacupuncture Therapy Mechanisms Explored
2
Immunofluorescent Labeling of Spheroids
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Spheroids were fixed with 4% paraformaldehyde for 24 h, dehydrated through a graded sucrose series, paraffin embedded, and sectioned. Four‐micron‐thick sections were blocked with 10% goat serum for 30 min at room temperature, and incubated with rabbit polyclonal anti‐DLL4 (diluted 1:1000; Abcam) and mouse monoclonal anti‐mucin 5AC (diluted 1:100; Santa Cruz) antibodies, respectively, for 1 h. After three washes with PBS, the sections were incubated with the corresponding secondary Alexa Fluor 488 or 594 secondary antibodies (all diluted 1:200, Invitrogen). Nuclei were counterstained with 4′,6‐diamidino‐2‐phenylindole (DAPI; Sigma‐Aldrich). Fluorescence was visualized on an immunofluorescence microscope (FV‐1000, Olympus, Japan). In all experiments, at least 10 spheroids per group were analyzed.
3
Western Blot Analysis of RPE-Choroid Proteins
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Whole protein extracts were isolated from isolated retinal pigment epithelium (RPE)-choroid tissues with RIPA buffer (Santa Cruz Biotechnology, Inc.) and were then quantified with Quick Start™ Bradford Protein Assay kit (Bio-Rad Laboratories, Inc.). Subsequently, 20 mg of protein extracts were resolved by 10% SDS-PAGE gel and transferred onto PVDF membranes (Immobilon™; Sigma-Aldrich; Merck KGaA). The membranes were blocked with 5% fat-free milk for 30 min at room temperature, probed for overnight at 4˚C with rabbit polyclonal anti-Dll4 (1:200; Abcam), goat polyclonal anti-delta-like 1 (Dll1; 1:200; cat. no. ab85346, Abcam), goat polyclonal anti-CD80 (1:500; cat. no. AF740, R&D Systems, Inc.), rabbit polyclonal anti-CD206 (1:500, cat. no. ab64693, Abcam), rabbit monoclonal anti-monocyte to macrophage differentiation associated (MMD) (1:200; cat. no. ab173967, Abcam), anti-β-actin antibody (1:2,000; cat. no. AC-15; Sigma-Aldrich; Merck KGaA), and then incubated with peroxidase-conjugated anti-rabbit-IgG (cat. no. A21020) or anti-goat-IgG (cat. no. A21030) (both 1:1,000; Abbkine Scientific Co., Ltd.) for 2 h at room temperature. The proteins were detected using ImmunoStar LD reagents (Wako Pure Chemical Industries, Ltd.) and visualized with a luminescent imager (Ez-Capture; ATTO Corporation).
4
Western Blot Analysis of Notch Signaling
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The protein samples were analyzed using a BCA kit (Pierce Company). The samples were separated by 10–20% Ready Gels (Bio-Rad, USA) and transferred onto a polyvinylidene fluoride (PVDF) membrane for 30 min in a transfer electricity meter (Bio-Rad, USA). The PVDF nitrocellulose films were cleared and sealed with 5% milk. After washing, anti-Notch 1 (1: 500, Abcam, USA), anti-Notch 2 (1: 800, Abcam, USA), anti-Notch 3 (1: 1000, Abcam, USA), and anti-DLL4 (1: 1000, Abcam, USA) were added to incubate overnight at 4°C. Then, these films were washed 3 times with Tris-buffered saline tween (TBST) and incubated with HRP secondary antibodies (1: 5000, Sigma, USA) at room temperature for 1 h. Relative expression levels of each protein were normalized to endogenous control β-actin using Image J software.
5
Western Blot Analysis of Cellular Proteins
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Cells were lysed in cell lysate, and then centrifuged at 12,000 × g for 20 min at 4 °C. The supernatant was collected and denatured. Proteins were separated in 10% SDS-PAGE and blotted onto polyvinylidene difluoride membrane (PVDF). The PVDF membrane was treated with TBST containing 50 g/L skimmed milk at room temperature for 4 h, followed by incubation with the primary antibodies: anti-CTR2 (1:200, Novusbio), anti-BCL-2 (1:500, Immunoway), anti-OCT-4 (1:1000, Proteintech), anti-KLF4 (1:500, Proteintech), anti-MDR1 (1:200, Santa),, anti-Notch1 ICD (1:1000, Abcam), anti-Notch2 ICD (1:1000, Abcam), anti-Jagged1 (1:500, Abcam), anti-Jagged2 (1:1000, Abcam), anti-DLL1 (1:500, Abcam), anti-DLL4 (1:500, Abcam), anti-Notch ICD (1:1000, Cell Signaling), anti-SDF-1 (1:1000, Abcam), anti-CXCR4 (1:2000, Abcam) and anti-β-actin (1:1000, Cell signaling) respectively, at 37 °C for 1 h. Membranes were rinsed and incubated for 1 h with the correspondent peroxidase-conjugated secondary antibodies. Chemiluminent detection was performed with the ECL kit (Pierce Chemical, Rockford, IL, USA). The amount of the protein of interest, expressed as arbitrary densitometric units, was normalized to the densitometric units of ß-actin.
6
HUVEC and Hindbrain Tissue Lysis
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HUVEC pellets were lysed in RIPA buffer (50 mM Tris-HCL at pH 7.4, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, 150 mM NaCl, 2 mM EDTA) supplemented with protease and phosphatase inhibitor cocktail (Roche). Hindbrains were lysed in Tris buffer (20 mM Tris at pH 9, 2% SDS) supplemented with protease and phosphatase inhibitor cocktail (Roche), boiled at 100°C, and then incubated at 750 rpm at 80°C. Insoluble material was removed, protein concentration was determined with the BCA protein assay kit (Thermo Scientific), and 10 μg of protein per lane was separated by SDS-PAGE. DLL4 was detected using anti-DLL4 (1:1000; Abcam, 7280) and membranes were reprobed with anti-β-Actin antibody ( 1:100,000; Abcam, clone AC-15). Band intensities of DLL4 and β-Actin were quantified with ImageLab software (Bio-Rad).
7
Protein Quantification and Western Blot Analysis
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Total protein concentrations of supernatant fractions were determined using the bicinchoninic acid (BCA) protein assay (Bio-Rad, Inc.). Equal amounts of protein aliquots were boiled in equal volumes of 2× SDS Laemmli sample buffer and resolved on 8% or 10% (w/v) with primary antibodies; anti-NOTCH1 (0.2μg/ml; Abcam®, Inc.), anti-Dll4 (0.1μg/ml; Abcam®, Inc.), anti-LYVE-1 (1μg/ml; Abcam®, Inc.) and anti-PECAM (2μg/ml; Abcam®, Inc.). Immunoreactive bands were detected with horseradish peroxidase-conjugated secondary antibodies and visualized using the enhanced chemiluminescence technique.
8
Western Blot Analysis of Notch Signaling
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Total proteins were extracted from cells and tissues using the T-PER Tissue Protein Extraction Reagent (ThermoFisher Scientific) with the Halt Protease & Phosphatase Inhibitor (ThermoFisher Scientific). Proteins were electrophoresed in 4–15% Tris-glycine sodium dodecyl sulfate-polyacrylamide gel (Bio-Rad Laboratories, Hercules, CA). Membranes were incubated with the following antibodies: human primary anti-Notch cleaved 1 (1:1000, Cell Signaling Technology, Pero, Italy); purified human anti-HES1 (1:1000, Cell Signaling Technology, Danvers, MA); anti-VEGFA (1:1000, Abcam, Cambridge, UK), anti-DLL4 (1:1000, Abcam, Cambridge, MA), anti-CD31 (1:1000, Abcam, Cambridge, MA), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:1000, Merck Millipore, Burlington, MA, USA). A secondary anti-rabbit or anti-mouse antibody (1:5000, Cell Signaling Technology, Danvers, MA) was used. Development was performed with a chemiluminescence system using the Clarity Max Western ECL Substrate (Bio-Rad Laboratories), and the signal displayed by the ChemiDoc MP instrument (Bio-Rad Laboratories) using Image Lab 5.2.1. The relative density of the bands was calculated using the Image J software.
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