Sunitinib and sorafenib were from LC Laboratories (Woburn, MA, USA). Sodium bicarbonate and HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) were from Sigma-Aldrich. Recombinant human VEGF was from Peprotech (#100-20-10). Cycloheximide was purchased from Santa Cruz biotechnology (#SC-3508). For Western blot analysis, the following primary antibodies and concentrations were used: anti-phospho-Akt antibody (1:500) (#4060; Cell Signaling Technology), anti-Akt antibody (1:1000) (#2920; Cell Signaling Technology), anti-VEGFR-2 antibody (1:1000) (#2479; Cell Signaling Technology), anti-VEGFR-1 (1:500) (#sc-316; Santa Cruz biotechnology), anti-β-actin antibody (1:5000) (#A2228; Sigma Aldrich), anti-Tie-2 antibody (1:500) (#MAB313; R&D systems) and anti-FGFR-1 antibody (1:1000) (#sc-121; Santa Cruz Biotechnology). Immunohistochemical staining were performed with anti-CD31 antibody (#Rb-10333-PO; Thermo Scientific). For immunofluorescence anti-CD31 (1:50) (# 553370; BD Pharmigen) and anti-VEGFR-2 antibody (1:50) (#2479; Cell Signaling Technology) were used. For flow cytometry, the following antibodies and dilutions were used: anti-CD31 (1:100) (#17-0319; eBioscience), anti-avb3 integrin (1:100) (MAB1976; Millipore), anti-VCAM-1(1:100) (#12-1069; eBioscience), anti-ICAM-1 (1:100) (#12-0549; eBioscience) and anti-MCP-1 (1:100) (#12-7099; eBioscience).
Anti icam 1
Manufactured by Thermo Fisher Scientific
Sourced in United States
Anti-ICAM-1 is a laboratory reagent used for the detection and quantification of intercellular adhesion molecule-1 (ICAM-1) in various sample types. It functions as a tool for researchers to investigate cell-cell interactions and inflammatory processes.
Automatically generated - may contain errors
Lab products found in correlation
Anti vcam 1, by Thermo Fisher Scientific (3 mentions)
Facscalibur, by BD (2 mentions)
Horseradish peroxidase conjugated anti rabbit or mouse igg secondary antibodies, by GeneTex (2 mentions)
Imagequant las 4000, by GE Healthcare (2 mentions)
Enhanced chemiluminescence solution, by Bio-Rad (2 mentions)
α tubulin, by GeneTex (2 mentions)
Las 4000, by Cytiva (2 mentions)
Anti cd31, by BD (1 mentions)
Anti cd31, by Thermo Fisher Scientific (1 mentions)
Anti tie2 antibody, by R&D Systems (1 mentions)
Anti vegfr 1, by Santa Cruz Biotechnology (1 mentions)
Anti fgfr 1 antibody, by Santa Cruz Biotechnology (1 mentions)
Anti mcp 1, by Thermo Fisher Scientific (1 mentions)
Recombinant human vegf, by Thermo Fisher Scientific (1 mentions)
Sodium bicarbonate, by Merck Group (1 mentions)
Anti cd31 antibody, by Thermo Fisher Scientific (1 mentions)
Anti phospho akt antibody, by Cell Signaling Technology (1 mentions)
Anti vegfr2 antibody, by Cell Signaling Technology (1 mentions)
Anti akt antibody, by Cell Signaling Technology (1 mentions)
Hepes, by Merck Group (1 mentions)
11 protocols using anti icam 1
1
Evaluation of Anti-Angiogenic Drug Effects
2
Immunophenotyping of Cell Populations
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Cells were stained with anti-CD40, anti-ICAM-1 (eBiosciences, San Diego, CA, USA), or isotype control mAbs. Cells were analyzed on an LSR II flow cytometer (Becton Dickinson, San Jose, CA, USA). FlowJo software version 10.10 (Tree Star Inc., Ashland, OR, USA) was used for analysis.
3
Validating Mast Cell Purity and EMT
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To validate the purity of isolated MCs, omentum-derived cells were verified by ICAM-1 expression (anti-ICAM-1; eBioscience, CA, USA), determined according to a previously published protocol [14 (link)]. To validate changes in the EMT process, phalloidin-labeled staining (Life Technologies, NY, USA) and α-SMA staining (Sigma-Aldrich) were carried out [30 (link)]. DAPI staining was conducted for visualization of cell nuclei (Sigma-Aldrich).
4
Quantification of Endothelial Activation Markers
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Immortalized HUVECtert cells (Schiller et al., 2009 (link)) were maintained in EBM™ growth medium supplemented with 10% FBS, EBM SingleQuots (Lonza, Basel, Switzerland), 100 U·mL−1 benzylpenicillin, 100 μg·mL−1 streptomycin and 1% amphotericin. The HUVECtert cell line was used instead of primary HUVEC in order to overcome batch-to-batch variability. The cells were seeded at a density of 5 × 105 cells per well overnight in 6-well plates. Then, cells were pretreated with plumericin for 30 min before TNF-α (10 ng·mL−1) stimulation for additional 14 h (VCAM-1 and ICAM-1 quantification) or 5 h (E-selectin). Cells were stained with FITC-labelled antibodies [anti-VCAM-1 (BD Biosciences, Schwechat, Austria; 551146), anti-ICAM-1 (eBioscience, Vienna, Austria; BMS108FI), anti-E-selectin (Bio-Rad AbD Serotec, Puchheim, Germany; MCA1969F)] and analysed with a FACSCalibur™ (BD Biosciences) flow cytometer.
5
Calpain Inhibition in Inflammatory Response
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Sodium taurocholate was obtained from Sigma (St. Louis, USA). Emodin was purchased from Dalian Food and Drug Administration (Dalian, China). The calpain inhibitor (PD150606) was obtained from Alexis Biochemicals (San Diego, USA). The amylase, TNF-α, and IL-6 assay kits were obtained from Lengton Bioscience Co. (Shanghai, China). 4% paraformaldehyde was purchased from Solarbio (Beijing, China). Dextran T500 was obtained from Sigma (St. Louis, USA). Anti-CD 2, anti-CD 5, and anti-CD 45R were purchased from PharMingen (San Diego, USA). Anti-F4/80 was purchased from AbD Serotec (Oxford, UK). Anti-ICAM-1 was purchased from eBioscience (San Diego, USA). Goat anti-rabbit IgG microbeads were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). The Fluo-3-AM assay kit was obtained from Dojindo Laboratories (Kyushu, Japan). Antibodies against calpain 1, caspase 12, caspase 3, β-tubulin, and goat anti rabbit IgG (H+L) secondary antibody were obtained from Abcam (Cambridge, UK). Calpain activity assay kit was obtained from Genmed (Boston, USA). The apoptosis detection kit (Annexin V-FITC/PI) was purchased from Solarbio (Beijing, China).
6
Cytokine Regulation of Endothelial Markers
7
Characterization of Mesothelial Cells
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The purity of the MC from effluents and omentum was determined by ICAM-1 expression (anti-ICAM-1; eBioscience, San Diego, CA), and subgroup classifications of MCs from effluents were determined by cytokeratin 18 expression (anti-cytokeratin 18, Santa Cruz Biotechnology, Santa Cruz, CA). Phalloidin-labeled staining was used to investigate the expression of MCs cytoskeletal actin as described previously [20 (link)].
8
Quantifying Cell Surface Expression
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To determine cell surface expression, 1x10 5 cells were cultured as indicated. Cells were stimulated with retinoic acid (100 nM), the agonistic anti-LTβR mAb or both for 24 hrs and 72 hrs. Cells were harvested by trypsinization and subsequently stained with biotin conjugated anti-VCAM-1 or anti-ICAM-1 (both eBioscience, Immunosource, Halle-Zoersel, Belgium), unlabeled anti-MAdCAM-1 (clone MECA 367, affinity purified from hybridoma cell culture supernatants) and unlabeled anti-LTβR mAb and subsequently counterstained with streptavidin-Alexa 488 and goat-anti-rat-Alexa 488 (all Invitrogen, Breda, The Netherlands), respectively. Sytox Blue (Invitrogen) staining was used to discriminate between live and dead cells. Data were acquired on a Cyan ADP High Performance Research Flow Cytometer (Beckman Coulter) and were analyzed with Summit Software v4.3.
For cell sorting, single cell suspensions of lymph nodes were stained in 100 µl diluted antibody mixtures containing the following antibodies; CD45-PE-Cy7 (Biolegend), Ter119-BV605 (Biolegend), GP38-Alexa Fluor 488 (Biolegend), CD31-PE (Biolegend), MHC-II-647(Clone M5-114, MO2Ab facility) and Cd11c Apc-Cy7 (Biolegend). Cells were sorted on a BD Fusion (BD Biosciences) using a 100 µm nozzle. Sorted cells were collected in ice-cold HBSS (Invitrogen), centrifuged and lysed in Trizol.
For cell sorting, single cell suspensions of lymph nodes were stained in 100 µl diluted antibody mixtures containing the following antibodies; CD45-PE-Cy7 (Biolegend), Ter119-BV605 (Biolegend), GP38-Alexa Fluor 488 (Biolegend), CD31-PE (Biolegend), MHC-II-647(Clone M5-114, MO2Ab facility) and Cd11c Apc-Cy7 (Biolegend). Cells were sorted on a BD Fusion (BD Biosciences) using a 100 µm nozzle. Sorted cells were collected in ice-cold HBSS (Invitrogen), centrifuged and lysed in Trizol.
9
Antibodies and Inhibitors for JAK-STAT Signaling
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Anti-p-JAK1 (Tyr1022/1023), p-JAK2 (Tyr1007/1008), p-FAK (Tyr397), and p-GSK3α/β (ser21/9) antibodies were purchased from Cell Signaling Technology (Danvers, MA, US). JAK1 and JAK2 were obtained from Elabscience (Houston, TX, US). U0126 was purchased from MCE (Monmouth Junction, NJ, US). JAK1 was purchased from Merck Millipore (Temecula, CA, US). Leptin was obtained from PeproTech (Rocky Hill, NJ, US). Anti-ICAM-1, ObR, FAK, p-ERK1/2, ERK, GSK3α/β antibodies, and ObR and control small interfering (si)RNAs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Stattic was purchased from Selleckchem (Houston, TX, US). AG490, PF573228, SB216763, anti-GAPDH, α-tubulin, and β-actin antibodies were obtained from Sigma-Aldrich (St. Louise, MO, US). Anti-p-GSK3β (Tyr216/279) and Anti-ICAM-1 antibodies were obtained from Thermo Scientific (Waltham, MA, US). Recombinant soluble ICAM-1 was purchased from KingFisher Biotech (Saint Paul, MN, US).
10
Western Blot Analysis of Orbital Tissue
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Lysates were gained from orbital tissues, which were injected with BSS (sham), hPMSCs, hPMSCsPRL-1, or steroids by homogenization with PRO-PREP solution (Intron, Gyeonggido, Korea). Equal concentration of protein were loaded by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to membranes. The membranes were incubated with anti-ICAM-1 (Thermo Fisher Scientific), TGFβ2 (GeneTex, Irvine, CA, USA), TSHR (NSJ Bioreagents, San Diego, CA, USA), or α-tubulin (GeneTex). After washing, it was incubated at room temperature for 2 h with horseradish peroxidase-conjugated anti-rabbit or mouse IgG secondary antibodies at a dilution of 1:5000 (GeneTex). The immune response bands were visualized with enhanced chemiluminescence solution (Bio-Rad Laboratories) and detected using an ImageQuant LAS 4000 (GE Healthcare Life Sciences, Little Chalfont, UK).
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