Spinal cord tissues were collected and washed with PBS, placed at 4∘ C, and homogenized using T 25 digital homogenizer (IKA, Seoul, Korea) in lysis buffer (1× RIPA lysis buffer) and then finally passed through a 311/2 gauge syringe needle and centrifuged at 14,000 rpm at 4∘C for 15 min. Protein concentration was determined in supernatants using Bio-Rad DC Protein Assay (Hercules, CA, USA). Equal amounts (40 μg) of protein were separated electrophoretically by 10% SDS-PAGE electrophoresis, and the resolved proteins were transferred to PVDF membranes (Millipore, Bedford, MA, USA). The membranes were incubated for 1 h with 5% non-fat skim milk prepared in TBS buffer to block nonspecific binding. The membranes were then incubated overnight with primary antibodies to ANGPT-1 (1:10000, Abcam, Cambridge, UK), ANGPT-2 (1:1000, Abcam), NE (1;1000), ZO-1 (1:500, Invitrogen, California, USA), AKT (1:1000, Cell Signaling Technology, Danvers, MA, USA), pAKT (1:1000, Cell Signaling Technology,) LC3B (1:1000, Cell Signaling Technology,), and Actin (1:10000, ABM). After 1-h incubation with corresponding secondary antibodies, The blots were visualized with a PowerOpti-ECL (Animal Genetics Inc., Gainesville, FL, USA) detection system, according to the recommended procedure. Immunoreactivity was detected using the BIORAD ChemiDoc™ XRS.
Angpt 1
Manufactured by Abcam
Sourced in United Kingdom, United States
ANGPT-1 is a protein that plays a key role in the regulation of angiogenesis, the process of new blood vessel formation. It is a member of the angiopoietin family of proteins and acts as a ligand for the Tie-2 receptor, which is expressed on endothelial cells. ANGPT-1 is involved in the stabilization and maturation of blood vessels.
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Lab products found in correlation
Angpt2, by Abcam (3 mentions)
Vegf a, by Merck Group (2 mentions)
α sma, by Abcam (2 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Gapdh, by Merck Group (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Occludin, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Abcam (1 mentions)
Fibroblast growth factor 2 (fgf2), by Abcam (1 mentions)
Vegfa, by Abcam (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Moma 2, by Bio-Rad (1 mentions)
5 protocols using angpt 1
1
Spinal Cord Protein Expression Analysis
2
Western Blot Analysis of Angiogenic Factors
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Proteins were isolated from treated RAW 264.7 cells and mice aortas. Tissues were lysed in lysis buffer (100 mM Tris-Cl, pH 6.8, 4%(m/v) SDS, 20% (v/v) glycerol, 200 mM β-mercaptoethanol, 1 mM PMSF, and 1 g/ml aprotinin) and proteins were transferred to PVDF membranes, which were then incubated with primary antibodies for VEGF-A (1:500, Millipore), ANGPT-1 (1:500, Abcam), ANGPT-2 (1:1000, Abcam), and GAPDH (1:5000, Sigma-Aldrich, Louis, USA) overnight at 4°C. The membrane bands were visualized by use of chemiluminescence (Millipore) and quantified by densitometry.
3
Evaluating BZA Effects on BSCB Permeability
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Immunocytochemistry was performed to evaluate the effects of BZA on BSCB permeability and angiogenesis. hCMEC/D3 cells were cultured on a coverslip in 24-well plate for 24 h and then fixed in a 4% paraformaldehyde. The cells were permeabilized using 0.5% Triton-X reagent and blocked with 5% BSA and 0.5% Tween-20 in PBS at room temperature for 1 h. Then, cells were incubated with primary antibodies overnight at 4 °C: zonula occludens-1 (ZO-1, 1:100, Invitrogen, Waltham, MA, USA, #40-2300) and occludin (1:100, Invitrogen, Waltham, MA, USA #33-1500), angiopoietin-1 (ANGPT-1, 1:200, Abcam, Cambridge, UK, ab133425), von Willebrand factor (vWF, 1:50, Millipore, Burlington, MA, USA, AB7356), and alpha smooth muscle actin (α-SMA, 1:200, Abcam, Cambridge, UK, ab21027). After that, the cells were incubated with secondary antibodies at a 1:500 ratio at room temperature for 1 h; donkey anti-rabbit Alexa Fluor®647 (Invitrogen, Waltham, MA, USA, A31573) and goat anti-mouse Alexa Fluor® 488 (Abcam, Cambridge, UK, ab150117). Lastly, the cells were mounted after 5 min of incubation in DAPI (1:500, Invitrogen, Waltham, MA, USA, D1356) and were analyzed using a fluorescence microscope (Zeiss 880, Carl Zeiss, Oberkochen, Germany) and ImageJ software (National Institutes of Health, Bethesda, MD, USA).
4
Quantifying Angiogenic Factors in BMSC Cultures
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The levels of VEGF-A, ANGPT-1 and FGF-2 (Abcam, USA) in supernatants of BMSCs cultured on both scaffolds were assessed using enzyme-linked immunosorbent assay (ELISA) kits. The supernatants were collected after 3 and 7 days of culturing and determined by ELISA as per the manufacturer’s instructions.
5
Histopathological Analysis of Atherosclerosis
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To assess overall burden and distribution of atherosclerosis, en face lesion staining with Oil-Red O was performed as previously described [15 , 42 (link)]. Cross-sections of the aortic roots (predilection site of atherosclerosis) were stained with hematoxylin and eosin (H&E) following a standard protocol of our lab. The content of lipids and collagen of aortic plaques was detected by Oil-Red O staining and Picrosirius red staining, respectively. The immunohistochemical staining procedure was as previously described [15 , 42 (link)]. Targeted proteins were identified by the following antibodies against macrophages/monocytes antigen (MOMA-2, 1:150, AbD Serotec, Oxford, UK), α smooth muscle actin (α-SMA, 1:200, Abcam, Cambridge, UK), ANGPT-1 (1:100, Abcam), ANGPT-2 (1:200, Abcam), and VEGF-A (1:150, Millipore).
Histological and immunohistochemical staining was analyzed by using Image-Pro Plus 6.0 (IPP 6.0, Media Cybernetics, MD, USA). The en face analysis of aortas was performed as previously described [15 , 42 (link)]. Plaque sizes were analyzed by H&E staining of the cross-sectional aortic sinus. Lipid deposition and content of collagen, macrophages, SMCs and angiogenic factors were analyzed as the ratio of the positive staining area of targeted protein to total plaque area. Fibrous cap thickness was evaluated by Picrosirius red staining as described [38 (link), 42 (link)].
Histological and immunohistochemical staining was analyzed by using Image-Pro Plus 6.0 (IPP 6.0, Media Cybernetics, MD, USA). The en face analysis of aortas was performed as previously described [15 , 42 (link)]. Plaque sizes were analyzed by H&E staining of the cross-sectional aortic sinus. Lipid deposition and content of collagen, macrophages, SMCs and angiogenic factors were analyzed as the ratio of the positive staining area of targeted protein to total plaque area. Fibrous cap thickness was evaluated by Picrosirius red staining as described [38 (link), 42 (link)].
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