Tissue sections were fixed with 4% PFA, permeabilized with 0.1% Triton X-100, and blocked with 3% BSA. Sections were incubated at 4 °C overnight in 0.3% BSA containing primary antibodies: FITC anti-α-smooth muscle actin (ab8211, Abcam; 1:500), anti-Ly6G (127602, BioLegend; 1:800), anti-MYH11 (ab125884, Abcam, 1:200), anti-Fibronectin-1 (sc-271098, Santa Cruz, 1:100), anti-CD36 (AF2519, R&D systems, 1:100), anti-KLF2 (NBP2-45510, Novus Biologicals, 1:100), anti-ACTC1 (MAB93081-SP, R&D systems, 1:100), or anti-COL1A1 (NB600-408, Novus Biologicals, 1:100). After several washes with PBS, sections were incubated with Alexa Fluor 488- or Alexa Fluor 594-conjugated secondary antibodies for 1 h at room temperature. Negative control slides were stained with secondary antibody only. TUNEL staining was performed on fresh frozen sections using an In Situ Cell Death Detection Kit (Roche, Catalog #12 156 792 910) according to manufacture instructions. Co-staining with FITC anti-α-smooth muscle actin (ab8211, Abcam; 1:500) was performed. DAPI-containing mounting media (GBI Labs, Catalog #E19-100) was used as a counterstain. Images were acquired with a Nikon A1RS confocal microscope system.
Ab8211
Manufactured by Abcam
Sourced in United Kingdom
Ab8211 is a primary antibody specifically targeting the protein of interest. It is suitable for use in various immunological applications such as Western blotting, immunohistochemistry, and immunocytochemistry. The antibody is purified and provided in a liquid format.
Automatically generated - may contain errors
Lab products found in correlation
Confocal microscope, by Nikon (3 mentions)
In situ cell death detection kit, by Roche (2 mentions)
Axiovert 200m, by Zeiss (2 mentions)
Ab6672, by Abcam (1 mentions)
Ab260043, by Abcam (1 mentions)
Ab6671, by Abcam (1 mentions)
Ab109725, by Abcam (1 mentions)
Ab34843, by Abcam (1 mentions)
Ab181602, by Abcam (1 mentions)
Bs 1374r, by Bioss Antibodies (1 mentions)
Bs 2973r, by Bioss Antibodies (1 mentions)
Bs 3418r, by Bioss Antibodies (1 mentions)
Ab192234, by Abcam (1 mentions)
Fv1000, by Olympus (1 mentions)
Embryoid body dissociation kit, by Miltenyi Biotec (1 mentions)
Permeabilization buffer, by BioLegend (1 mentions)
Alexa 647, by Thermo Fisher Scientific (1 mentions)
Mab3715, by R&D Systems (1 mentions)
Sc 3877, by Santa Cruz Biotechnology (1 mentions)
Facsuite software, by BD (1 mentions)
12 protocols using ab8211
1
Multiparametric Immunofluorescence Analysis
2
Macrophage Polarization and Skin Fibrosis
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Western blotting was performed to explore M2 polarization of macrophages and the level of skin fibrosis. The following antibodies were used in the western blotting assays: alpha-smooth muscle actin (α-SMA, 1:100, ab8211, Abcam); IL-10 (1:100, ab34843, Abcam); type I collagen (Col Ⅰ, 1:200, ab260043, Abcam); IL-6 (1:500, ab6672, Abcam); cytokeratin 17 (1:500, ab109725, Abcam); TNF-α (1:1000, ab6671, Abcam); arginase-1 (1:500/1:50, #93668, Cell Signaling Technology); CD206 (1:1000, #PA5-114310, Invitrogen); Smad1/5 (1:500, bs-2973R, Bioss Antibodies); phospho-Smad1/5 (1:1000, bs-3418R, Bioss Antibodies); BMP4 (1:500, bs-1374R, Bioss Antibodies); and GAPDH (1:10000, ab181602, Abcam).
3
Immunofluorescence Assay of Syne1 in MC3T3-E1 Cells
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MC3T3-E1 cells were washed in PBS and fixed in 4% paraformaldehyde for 15 min. Cells were permeabilized with Triton X-100 (0.025%) for 10 min, blocked in 1% goat serum for 1 h and probed with primary antibody anti-Syne1 (1:100; ab192234, Abcam) at 4 °C overnight. Cells were washed and labeled with FITC-conjugated secondary antibody (ab8211, Abcam) for 1 h. Nuclei were stained with DAPI for 10 min and imaged on a confocal microscope (FV1000, Olympus, Japan).
4
Differentiation of Adipocytes and SMCs in EBs
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To determine percentage differentiation of adipocytes and SMCs following EB development, cells from D3 and HO-1–/– 7-day EBs were subjected to flow cytometric analysis. EBs were first dissociated by Embryoid Body Dissociation kit (Miltenyi Biotec, Germany), followed by filtering through 70-µm strainers (MACS SmartStrainers) to obtain single cell suspensions. To detect adipocytes, cells were stained with Nile red as described [45] . Briefly, cells were suspended in ice-cold PBS and Nile red (Sigma, stock solution 100 µg/mL in DMSO) added to a final concentration of 0.1 µg/mL, incubated for 5 min, centrifuged, and washed once with PBS. Afterwards, cells were resuspended in an appropriate volume of PBS and kept on ice prior to flow cytometric analysis. To detect SMCs, cells were washed, fixed with 4% paraformaldehyde, permeabolized with 0.1% Triton X-100, blocked with 5% BSA, and then stained with anti-alpha smooth muscle actin antibody-FITC (Abcam, ab8211) or isotype control IgG2a-FITC (Abcam, ab18455). Acquisition was performed using FACS Calibur and cells analyzed using FlowJo software.
5
Fibroblast Phenotype Characterization
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Fibroblasts were washed with PBS and dissociated from the vessels with trypsin. The cells were fixed with 4% paraformaldehyde at 37 °C for 10 min. The cells were incubated with 1:100 dilution of primary antibody against FAP (MAB3715, R&D Systems) or IgG1 (sc-3877, Santa Cruz Biotechnology, Dallas, TX, USA) for 30 min at room temperature and incubated with 1:200 dilution of secondary antibody Alexa647 (A21237, Thermo Fisher Scientific) for 30 min at room temperature in the dark. The cells were permeabilized with a permeabilization buffer (#421002, BioLegend, San Diego, CA, USA) and a 1:100 dilution of antibody against α-SMA conjugated with FITC (ab8211, Abcam, UK) or IgG2a conjugated with FITC (#400210, BioLegend) for 30 min at room temperature in the dark. The cells were analyzed for two antigens simultaneously using a BD FACSLyric flow cytometer (BD Biosciences, San Jose, CA, USA), and the obtained data were estimated using BD FACSuite software (BD Biosciences). The analyzed cells were confirmed to exclude dead cells using Zombie Aqua (#423101, BioLegend).
6
Quantifying Apoptotic Cells in Venous Thrombosis
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Two hours before euthanasia, an intraperitoneal injection of propidium iodide (PI) (P1470 Millipore Sigma, Burlington, MA) dissolved in phosphate-buffered saline was delivered to mice at a dose of 15 mg/kg body weight, as described by Wang et al.13 (link) Mice were monitored for adverse reactions between administration and euthanasia. All mice tolerated treatment well with 0% mortality. At time of euthanasia, mice were perfusion fixed (4% paraformaldehyde) and IVC/thrombus samples were harvested and embedded in optimal cutting temperature compound for frozen sectioning. We cut 6-μm-thick sections, counterstained them with DAPI (E19-100; GBI Labs, Bothell, WA) and an antibody against α-smooth muscle actin (α-SMA) (ab 8211, Abcam, Cambridge, UK; 1 ng/μL). Sections were visualized on a Nikon Confocal Microscope. PI-positive cells, as compared with total nucleated cells, within the vein wall and thrombus were quantified across three to six high-powered images (fewer images were used in sham sections owing to less wall distension).
7
Immunostaining Analysis of Thrombus Samples
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Fresh frozen ICV/thrombus samples were cross-sectioned at a thickness of 6 μm. Sections were immunostained with the following primary antibodies: anti-CD31 (AF3628, R&D Systems, Minneapolis, MN; 1:40); anti-α-SMA (ab 8211, Abcam; 1:500); anti-RIPK3 (95702, Cell Signaling Technology, 1:100); anti pMLKL (phospho S345) (ab196436, Abcam, 1:100), anti-CD41 (MA5-16875, Invitrogen, Waltham, MA; 1:1000). Tissue samples were subsequently stained with AlexaFluor 488 and 594 antibodies. Sections were counterstained with DAPI-containing mounting media (Fluorescent Mounting Medium with DAPI, GBI Labs, Cat No E19-100/E19-18). Images were acquired on a Nikon Confocal Microscope. Quantification was performed using Fiji/Image J software: images were subject to uniform color thresholds to identify stained regions of interest and were subsequently processed to be binary. Total region for analysis was mapped using the polygon selection tool, and percent area positive for the stain of interest was subsequently measured. Three to five high-powered fields were analyzed per mouse.
8
TUNEL Assay for Apoptosis Quantification
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Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed on 6-μm IVC/thrombus fresh frozen sections using an In Situ Cell Death Detection Kit (Roche Holding AG, Basel, Switzerland; Cat No 12 156 792 910) according to manufacture instructions. Costaining with anti-α-SMA (ab 8211, Abcam; 1:500) was performed. Images were acquired on a Nikon Confocal Microscope. TUNEL positive cells, as compared with total nucleated cells, within the vein wall and thrombus were quantified across three to six high-powered images (fewer images were used in sham sections owing to less wall distension).
9
Western Blot Analysis of VSMC Markers
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The protein samples were extracted from VSMCs with RIPA lysis buffer (Beyotime, Haimen, China) on ice for 30 min. The process of western blot was used as previously described [37 (link)]. The following primary antibodies were used: anti-total ERK1/2 antibody (#9926, Cell signaling technology), anti-phospho-ERK1/2 antibody (#9910, Cell signaling technology), anti-GAPDH antibody (#2118, Cell signaling technology), anti-BMP2 antibody (sc-6895, Santa Cruz), anti-Runx2 antibody (sc-10758, Santa Cruz), anti-osteopontin antibody (ab8448, Abcam), anti-α-SMA antibody (ab8211, Abcam), anti-SM22α antibody (ab10135, Abcam). The bands were analyzed semi-quantitatively.
10
Quantifying Vascular αSMA Density
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Immunofluorescence staining of the tissue sections was performed using ASMA (1:100; ab8211, Abcam, Cambridge, MA, USA, and images were captured using an Axioscan Z1 whole slide scanner (Zeiss, Jena, Germany). Pictures were evaluated using Zen 3.3 Blue Edition (Zeiss Microscopy, Jena, Germany). The fluorescence channel for the α-smooth muscle actin (αSMA) was dimmed to enhance the blood vessel signal, and the density of the αSMA per mm² was measured using the ImageJ software, version 1.53e (developed by Wayne Rasband, National Institutes of Health, Bethesda, MD, USA), and Java 1.6.0_24 (64 bits). Counts matching 0.3 to 1.0 circularity were considered during our analysis.
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