Mouse anti icam 1

Manufactured by Santa Cruz Biotechnology

Sourced in Canada, United States

Mouse anti-ICAM-1 is an antibody that specifically binds to the Intercellular Adhesion Molecule 1 (ICAM-1) protein. ICAM-1 is a cell surface glycoprotein that plays a role in cell-cell adhesion and immune response.

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8 protocols using mouse anti icam 1

Immunohistochemical Analysis of Lung Tissue

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Paraffin-embedded lung sections (4-µm thickness) were used for this study. Images were taken under polarized light using an upright dry 40× objective. Tissues were deparaffinized and after antigen retrieval, permeabilized with 0.4% triton X-100. Tissues were blocked with 10% BSA and maintained in PBS + 5% BSA + 0.4% triton X-100 throughout antibody treatments. Primary antibodies were incubated at 4°C overnight and secondary antibodies were incubated for 1 hr at room temperature. Primary antibodies used (1:200 dilution) include rabbit anti-NFκB p65 (#8242, Cells Signaling Technology); mouse anti-cRel (#MA5-15859, Thermo Fisher Scientific); rabbit anti-NFκB1 (#13586, Cells Signaling Technology); mouse anti-NFκB1 (#NBP2-66976, Novus); rabbit anti-RUNX1(#ab229482, Abcam); goat anti-IL33 (#AF3626 R&D); mouse anti-ICAM1 (#sc-1511, Santa Cruz Biotechnology, Inc); and mouse anti-CD3 ((# sc-7296, Santa Cruz Biotechnology, Inc). Goat anti-rabbit and anti-mouse IgG-A594 or IgG-A488 were used as secondary antibodies (1:200 dilution at RT). For anti-goat, rabbit anti-goat IgGA-647 was used as secondary antibody (1:200 dilution at RT). DAPI was used for nuclear counterstaining. The ProLong Gold antifade reagent was used as mounting media. Mount slides were examined with a Leica DM6000 B. Slide Book 6 (3i) was used for analysis and capturing images.

Verma M., Verma D., Sripada A.S., Sirohi K., Varma R., Sahu A, & Alam R. (2023). NFκB1 inhibits memory formation and supports effector function of ILC2s in memory-driven asthma. Frontiers in Immunology, 14, 1217776.

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Western Blot Analysis of Endothelial Adhesion Molecules

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Cells were lysed with lysis buffer (Cell Signaling Technology, Beverly, MA, USA), and protein concentration was determined by Bradford assay (Bio-Rad, Hercules, CA, USA). Protein lysates (20 μg per lane) were separated in precast 4-12% Tris-glycine SDS-polyacrylamide gels (Invitrogen, Grand Island, NY), and proteins were transferred to PVDF membranes (Invitrogen, Grand Island, NY ). After blocking with 5% non-fat milk, PVDF membranes were incubated overnight at 4 °C with the following primary antibodies: rabbit anti-E-selectin (1:1000 dilution; Santa Cruz, Dallas, TX), rabbit anti-VCAM-1 (1:1000 dilution; Santa Cruz, Dallas, TX), mouse anti-ICAM-1 (1:1000 dilution; Santa Cruz, Dallas, TX). After washing, the membrane was incubated with peroxidase-conjugated secondary antibody at room temperature for 1 hour. Antigen–antibody complexes were visualized with the ECL chemiluminescence system (Amersham, Piscataway, NJ) and exposed to a Kodak X-OMAT film (Kodak, Rochester, NY, USA). The relative densities of bands were analyzed with NIH Image J.

Wu L., Walas S., Leung W., Sykes D.B., Wu J., Lo E.H, & Lok J. (2014). Neuregulin1-β decreases IL-1β-induced neutrophil adhesion to human brain microvascular endothelial cells. Translational stroke research, 6(2), 116-124.

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Immunoblotting of Endothelial Cell Proteins

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Cells were harvested with RIPA protein extraction reagent (Sigma-Aldrich) supplemented with protease inhibitors, and protein concentration determined using the Bradford reagent (Sigma-Aldrich). 30 μg of each sample were separated by SDS-PAGE and transferred to nitrocellulose membranes (Hybond ECL, Amersham Biosciences Europe GmbH, Milan, Italy). Membranes were blocked with Odyssey blocking buffer (LI-COR Biotechnology GmbH, Bad Homburg, Germany) diluted 1:1 with PBS for 30 min and probed with the following primary antibodies overnight: rabbit anti-Claudin-5 (1:200, Invitrogen, Cat. #34-1600, Lot. #RB232835), mouse anti-ICAM-1 (1:800, SantaCruz Biotechnologies, Santa Cruz, CA Cat. #sc-8439, Lot. #B2316), rabbit anti-VE-Cadherin (1:1000, Cell signaling, Cat. # 2500, Lot #D87F2), mouse anti-GAPDH (1:800, Millipore, Cat. #MAB374, Lot #2742734), rabbit anti-β-actin (1:1000, Sigma-Aldrich, Cat. #A2066, Lot #095M46V). Membranes were then processed for immunodetection using specific fluorescent IRDye^®680- or IRDye^®800-conjugated secondary antibodies (LI-COR, Cat. # 926-32211, Lot #C20906-02 and Cat. #926-68070, Lot #C20925-04). Detection of specific bands was carried out using the LI-COR Odyssey^® Infrared Imaging System (LI-COR Bioscience). Band intensity was analyzed using the image processing software “Image J” developed by NIH and in public domain.

Spampinato S.F., Merlo S., Fagone E., Fruciano M., Barbagallo C., Kanda T., Sano Y., Purrello M., Vancheri C., Ragusa M, & Sortino M.A. (2019). Astrocytes Modify Migration of PBMCs Induced by β-Amyloid in a Blood-Brain Barrier in vitro Model. Frontiers in Cellular Neuroscience, 13, 337.

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Quantifying Cell Adhesion Molecules

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Whole‐cell lysates were prepared in modified RIPA buffer (50 mmol/L of HEPES, 20 mmol/L of pyrophosphate, 25 mmol/L of β‐glycerophosphate, 50 mmol/L of NaF, 5 mmol/L of Na₂MoO₄, 5 mmol/L of EDTA, 150 mmol/L of orthophenanthrol, 1% NP‐40, 2% deoxycholate, and 1% Triton X‐100) in the presence of protease and phosphatase inhibitors. Protein concentrations were quantified using the Pierce BCA Protein Assay (Thermo Scientific, Waltham, MA). Samples (25 μg protein) were denatured, resolved on an 8% SDS‐polyacrylamide gel, and then transferred to an Immuno‐blot PVDF membrane (Amersham Biosciences, Piscataway, NJ). Incubation with primary antibodies (rabbit anti‐VCAM‐1, mouse anti‐ICAM‐1, rabbit anti‐E‐Selectin, and mouse anti‐GAPDH; Santa Cruz Biotechnology, Santa Cruz, CA) was performed for 2 hours, followed by HRP‐linked anti‐rabbit IgG or anti‐mouse IgG (Bio‐Rad, Hercules, CA) secondary antibodies for 40 minutes at room temperature. Immunoreactive bands were visualized by chemiluminescence using an Amersham ECL Prime Western Blotting Detection Kit (GE Healthcare, Pittsburgh, PA). Densitometry was performed using ChemiDoc MP System software (Bio‐Rad).

Vogel M.E., Idelman G., Konaniah E.S, & Zucker S.D. (2017). Bilirubin Prevents Atherosclerotic Lesion Formation in Low‐Density Lipoprotein Receptor‐Deficient Mice by Inhibiting Endothelial VCAM‐1 and ICAM‐1 Signaling. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 6(4), e004820.

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Antibody Panel for Molecular Markers

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Mouse anti-ICAM-1(Cat#: sc107), VCAM-1(Cat#: sc13160), GAPDH (Cat#: sc365062) SRA (Cat#: sc56777), αSMA (Cat#: sc130617), CD36 (Cat#: sc70644), and CD68 (Cat#: sc20060) monoclonal antibodies were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA). Rabbit anti-ABCG1 (Cat#: NB400-132), ABCA1(Cat#: NB400-105) and SRBI (Cat#: NB400-104) polyclonal antibodies were purchased from Novus Biologicals (Littleton, CO). Mouse anti-IL-1β (Cat#: #12242) monoclonal antibody was purchased from Cell Signaling Technology. Mouse anti-Arg (Cat#: ab239731) monoclonal antibody were purchased from Abcam (Cambridge, MA). Mouse anti-rabbit IgG-R (Cat#: sc2492), mouse anti-rabbit IgG-FITC (Cat#: sc2359) and m-IgGκ BP-FITC (Cat#: sc516140) antibodies were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA). Astragalus flavone (Cat#: SA9780) was purchased from Solarbio (Beijing, China).

Ma C., Zhang J., Yang S., Hua Y., Su J., Shang Y., Wang Z., Feng K., Zhang J., Yang X., Zhang H., Mao J, & Fan G. (2020). Astragalus Flavone Ameliorates Atherosclerosis and Hepatic Steatosis Via Inhibiting Lipid-Disorder and Inflammation in apoE−/− Mice. Frontiers in Pharmacology, 11, 610550.

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Quantifying Protein Expression in Cardiac Cells

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Intercellular adhesion molecule 1 (ICAM1) and vascular cell adhesion molecule 1 (VCAM1) protein levels were evaluated by SDS‐PAGE using goat anti‐ICAM1 (R&D Systems; catalog number AF‐796) and rabbit anti‐VCAM1 (Abcam; catalog number ab134047) antibodies, respectively. ATPase sarcoplasmic/endoplasmic reticulum Ca2⁺ transporting 2 (ATP2A2) protein level was evaluated by SDS‐PAGE using rabbit anti‐ATP2A2 antibodies (Badrilla; catalog number A010‐80). Phospholamban phosphorylation at serine 16 and threonine 17 was evaluated by SDS‐PAGE using rabbit anti–phosphorylated phospholamban serine 16 (Badrilla; catalog number A010‐12), rabbit anti–phosphorylated phospholamban threonine 17 (Badrilla; catalog number A010‐13), and mouse anti–total phospholamban (Badrilla; catalog number A010‐14). Ryanodine receptor 2 phosphorylation was evaluated by SDS‐PAGE using rabbit anti–phosphorylated ryanodine receptor 2 antibodies (Badrilla; catalog number A010‐31).
Human cadherin‐5 and ICAM1 protein levels were evaluated by SDS‐PAGE using rabbit anti–cadherin‐5 (Cell Signaling Technology; catalog number 2500) and mouse anti‐ICAM1 (Santa Cruz Biotechnology; catalog number sc‐8439) antibodies, respectively.
Protein loading quantity was controlled using mouse monoclonal anti–α‐tubulin antibodies (Sigma; catalog number T5168).

Cornuault L., Hérion F., Bourguignon C., Rouault P., Foussard N., Alzieu P., Chapouly C., Gadeau A., Couffinhal T, & Renault M. (2023). Partial Mural Cell Ablation Disrupts Coronary Vasculature Integrity and Induces Systolic Dysfunction. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 12(13), e029279.

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Phytochemical Analysis and Anti-inflammatory Effects

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Ferulic acid and 5-hydroxymethyl-2-furaldehyde (5-HMF) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Albiflorin and paeoniflorin were the products of Wako (Osaka, Japan). Nodakenin was purchased from NPC BioTechnology Inc. (Daejeon, Korea). The purity of all reference standards was ≥98.0%. HPLC-grade methanol, acetonitrile, and water were obtained from J.T.Baker (Phillipsburg, NJ, USA). Glacial acetic acid, analytical reagent grade, was purchased from Junsei (Tokyo, Japan). RPMI 1640, fetal bovine serum, TNF-α, tissue culture reagents, 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxy-methylester (BCECF-AM), DAF-FM diacetate, and CM-H₂DCFDA, Alexa Fluor 488 and 594 conjugated second antibodies were purchased from Invitrogen (San Diego, CA). Biotin 3′ End DNA Labeling Kit, LightShift® Chemiluminescent EMSA Kit, Biodyne® Precut Nylon Membranes, Lipofectamine LTX reagent, and Renilla-Firefly Luciferase Dual Assay Kit were purchased from Pierce Biotechnology (Rockford, USA). Primary antibodies, including mouse anti-ICAM-1, goat anti-VCAM-1, rabbit anti-E-selectin, mouse anti-NF-κB, mouse anti-p-IκB-α, rabbit anti-HO-1, and rabbit anti-Nrf2, were purchased from Santa Cruz Biotechnology (CA, USA). Donkey anti-goat IgG-H+I were purchased from Bethyl (Montgomery, USA) and goat anti-rabbit IgG and goat anti-mouse IgG were purchased from Enzo (Farmingdale, USA).

Choi E.S., Lee Y.J., Seo C.S., Yoon J.J., Han B.H., Park M.C., Kang D.G, & Lee H.S. (2016). Vascular Protective Role of Samul-Tang in HUVECs: Involvement of Nrf2/HO-1 and NO. Evidence-based Complementary and Alternative Medicine : eCAM, 2016, 9580234.

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Immunodetection Antibodies and Astragalus

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Mouse anti-ICAM-1(Cat#: sc107), VCAM-1(Cat#: sc13160), GAPDH (Cat#: sc365062) SRA (Cat#: sc56777), αSMA (Cat#: sc130617), CD36 (Cat#: sc70644), and CD68 (Cat#: sc20060) monoclonal antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Rabbit anti-ABCG1 (Cat#: NB400-132), ABCA1(Cat#: NB400-105) and SRBI (Cat#: NB400-104) polyclonal antibodies were purchased from Novus Biologicals (Littleton, CO). Mouse anti-IL-1β (Cat#: #12242) monoclonal antibody was purchased from Cell Signaling Technology. Mouse anti-Arg (Cat#: ab239731) monoclonal antibody were purchased from Abcam (Cambridge, MA). Mouse anti-rabbit IgG-R (Cat#: sc2492), mouse anti-rabbit IgG-FITC (Cat#: sc2359) and m-IgGκ BP-FITC (Cat#: sc516140) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Astragalus avone (Cat#: SA9780) was purchased from Solarbio (Beijing, China).

Ma C., Zhang J., Yang S., Hua Y., Su J., Shang Y., Wang Z., Feng K., Zhang J., Zhang H., Zhao L., Mao J., & Fan G. (2020). Astragalus flavone ameliorates atherosclerosis and hepatic steatosis via improving lipid metabolism and inhibition of inflammation in apoE-/- mice.

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