For Western blot, mice were perfused transcardially with PBS to remove blood. The brains were collected and different brain regions were dissected on ice. All the samples were stored at −80 °C. The brainstem samples were homogenized in cold RIPA lysis buffer containing proteinase and phosphatase inhibitors.28 (link),36 (link) Equivalent amounts of protein from each group were added to a 4–12% Bis-Tris-polyacrylamide electrophoresis gel. Then, the proteins were transferred to polyvinylidene fluoride (PVDF) membranes. We blocked the membranes with 5% skim milk and then incubated them with primary antibodies against TH (1:1000, ab129991, Abcam, Cambridge, MA, USA), Iba-1 (1:1000, 019–19,741, Abcam, Cambridge, MA, USA), CR3 (1:1000, ab128797, Abcam, Cambridge, MA, USA) or GAPDH (1:5000, ab181602, Abcam, Cambridge, MA, USA) at 4 °C overnight. The membranes were then washed three times with PBST and incubated with horseradish peroxidase-linked anti-rabbit IgG (1:3000) antibody at room temperature for 2 h. The blot signals were detected by ECL reagents (Biological Industries, Cromwell, CT, USA). The densitometry of blots was quantified based on previous report.46 (link) GAPDH was used as an internal control for each blotting. All blots were normalized to GAPDH. Fold changes for each treatment were normalized and are shown as percentages of the control.
Ab128797
Manufactured by Abcam
Sourced in United States
Ab128797 is a laboratory reagent manufactured by Abcam. It serves as a tool for scientific research, but its specific core function is not available in this factual, unbiased description.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ab129991, by Abcam (1 mentions)
Ab181602, by Abcam (1 mentions)
Ecl reagent, by Sartorius (1 mentions)
Ab15270, by Abcam (1 mentions)
Ab177487, by Abcam (1 mentions)
Ab178847, by Abcam (1 mentions)
Ab7260, by Abcam (1 mentions)
Dylight 549 donkey anti rabbit, by Thermo Fisher Scientific (1 mentions)
Alexa 488 conjugated anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Facscalibur flow cytometer, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Ab8226, by Abcam (1 mentions)
Ab1384, by Abcam (1 mentions)
Ab32503, by Abcam (1 mentions)
Ab9485, by Abcam (1 mentions)
Ab208670, by Abcam (1 mentions)
Cocktail of protease inhibitor, by Roche (1 mentions)
Ab157107, by Abcam (1 mentions)
Ab108327, by Abcam (1 mentions)
7 protocols using ab128797
1
Western Blotting Analysis of Brain Samples
2
Immunostaining Protocol for Cell Characterization
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Upon reaching 40%‐50% confluence, cells in each well were washed 3 times with pre‐cooled PBS (5 minutes each) and fixed with 1 mL of 95% pre‐cooled ethanol at −20°C for 30 minutes. The 95% ethanol was then removed and 1 mL of PBS containing 5% BSA was added for incubation for 60 minutes at room temperature. Then, the cells were incubated overnight at 4°C with 200 μL rabbit polyclonal antibody to CD11b (ab128797, 1 μg/mL, Abcam), rabbit monoclonal antibody to Iba‐1 (ab178847, 1:100, Abcam), rabbit monoclonal antibody to NeuN (ab177487, 1:300, Abcam), rabbit polyclonal antibody to GFAP (ab7260, 1:1000, Abcam) or rabbit polyclonal antibody to CC1 (ab15270, 1:800, Abcam) or rabbit IgG (serving as NC). Thereafter, the cells were incubated with DyLight 549‐donkey anti‐rabbit (SA5‐10064, Invitrogen) at room temperature for 2 hours without light exposure, mounted with glycerine and observed under a laser confocal microscope.
3
Microglia Isolation and Betanin Treatment
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Microglia primary culture was obtained from the cerebral cortex brains of 1–3 days old Wistar rat. Concisely, the mix glial cells were cultured for two weeks in DMEM high glucose, supplemented with 10% FBS. Microglial cells were isolated with a Shaker at 80 rpm for 30 min at 37°C. Harvested microglial cells were placed into 96-well plates at a density of 3×104 cells/well [24 (link)]. To confirm the purity of the harvested microglial cells, anti-CD11b antibody (ab128797, 1:200; Abcam), a typical microglia marker was used on FACS Calibur flow cytometer instrument and the secondary antibody was used Alexa-488 conjugated anti-rabbit IgG (A11008, Invitrogen).
Treatment of microglia cells was performed in three timepoints 24, 48 and 72h; each timepoint consist of the control group, without LPS and betanin, induced group with 1μg/ml LPS [24 (link)] and a group with 5 different doses of betanin, contains 100, 200, 300, 400 and 500μM which were selected for viability assay.
Treatment of microglia cells was performed in three timepoints 24, 48 and 72h; each timepoint consist of the control group, without LPS and betanin, induced group with 1μg/ml LPS [24 (link)] and a group with 5 different doses of betanin, contains 100, 200, 300, 400 and 500μM which were selected for viability assay.
4
Western Blot Analysis of Aortic and Macrophage Proteins
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Human and mouse aortic tissues or BMDMs were lysed in radioimmunoprecipitation buffer. The protein concentrations were evaluated using a BCA Protein Assay Kit (Thermo Scientific, Waltham, MA). Equal amounts of total protein were separated using 10% SDS‐PAGE and transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA). After blocking, the membranes were probed with primary antibodies against CD11b (ab128797, Abcam), p53 (ab26, Abcam), caspase3 (ab1384, Abcam), Bax (ab32503, Abcam), Akt (4691, Cell Signaling Technology, Beverly, MA), p‐Akt (4060, Cell Signaling Technology), β‐actin (ab8226, Abcam), and GAPDH (ab9485, Abcam) at 4℃ overnight. Then, the membranes were incubated with the appropriate horseradish peroxidase–conjugated secondary antibodies. The protein bands were detected by a chemiluminescence detection kit, and β‐actin or GAPDH was used as a loading control. All the experiments were independently conducted in triplicate.
5
Immunohistochemical Localization of Immune Cells
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Thin-sliced paraffin-embedded histological sections were processed with antigen retrieval procedures after deparaffinization. To reduce nonspecific binding, sections were incubated with 10% normal bovine serum at room temperature. To stain for macrophages or neutrophil leukocytes, sections were incubated with antibodies against CD11b (#ab128797, Abcam) or MPO (#ab208670, Abcam) at 4°C overnight. After washing with PBS, the tissues were incubated with HRP-labeled polymer anti-mouse antibodies (Dako, K4007) for 30 min at room temperature, and the conjugates were detected using Histofine Simple Stain AEC solution (#H1506, Nichirei Corporation, Tokyo, Japan). Sections were counterstained with hematoxylin. Immunofluorescence double staining for the histological sections was performed to examine the localization of NETs in the atherosclerotic plaques using the same protocol of immunocytochemistry described above. Specimens were observed under confocal microscopy.
6
Western Blot Analysis of Retinal Proteins
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Total protein was extracted from rat retina or Müller cells using RIPA lysis buffer (Sigma-Aldrich) with a cocktail of protease inhibitors (Roche Applied Science, Penzberg, Germany) following the manufacturer’s instructions. The protein expression level was determined by densitometric analysis and normalized to the level of GAPDH. Since different proteins with similar molecular weights were sourced from different gels and PVDF membranes, a separate GAPDH control was run for each blot and one of them was chosen for presentation. The primary antibodies were as follows: GAPDH (1:10000 dilution; Abcam, ab8245), CD11b (1:1000 dilution; Abcam, ab128797), CD68 (1:1000 dilution; Abcam, ab125212), Iba-1 (1:1000 dilution; Abcam, ab178846), OPN (1:1000 dilution; Abcam, ab8448), CD44 (1:5000 dilution; Abcam, ab157107), Itgαvβ3 (1:5000 dilution; Abcam, ab179475), P62 (1:1000 dilution; Cell Signaling Technology, 23214), LC3B (1:1000 dilution; Cell Signaling Technology, 12741), Atg5 (1:5000 dilution; Abcam, ab108327), Atg12 (1:1000 dilution; Abcam, ab303488), Beclin1 (1:1000 dilution; Cell Signaling Technology, 3495).
7
Immunofluorescence Staining of Aortic Tissues
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Serial paraffin sections from human and mouse aortic tissues were deparaffinized and rehydrated as previously described.21 Antigen retrieval was performed by boiling in 10 mmol/L Tris, 1 mmol/L, and 0.05% Tween20 retrieval buffer. The adherent BMDMs were fixed with 4% paraformaldehyde and permeabilized with 0.3% Triton X‐100 in PBS for 10 minutes. After blocking with 1% bovine serum in PBS at room temperature for 1 hour, the sections or slides were incubated with diluted primary antibodies against CD11b (ab128797, Abcam), MOMA‐2 (MCA519GT, Bio‐Rad), F4/80 (ab111101, Abcam), iNOS (ab15323, Abcam), and CD206 (PA5‐101657, Invitrogen) at 4°C overnight. Secondary fluorescent antibodies (Alexa Fluor 488, green; Alexa Fluor 568, red) were added and incubated at room temperature in the dark. D4',6‐diamidino‐2‐phenylindole was used for the immunofluorescence staining of cellular nuclei. The fluorescence signals were visualized by a fluorescence laser scanning confocal microscope (Leica Imaging Systems, Cambridge, UK).
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