Antibodies (abs) used were purified immunoglobulin [Ig] unless otherwise noted. Monoclonal anti-human Abs included the following: anti-glycophorin C and A produced in our laboratory [25] (link); anti-LW (BS46, generously provided by Dr. Jean-Pierre Cartron, INSERM Unité 665, Paris, France) [26] (link); anti-CD47 (6H9)[27] (link); anti-CD44 (Sigma-Aldrich, St. Louis, MO); anti-3-nitrotyrosine (Santa Cruz biotechnology, CA). Polyclonal anti-human Abs included the following: anti-mitogen-activated protein kinase (MAPK) ERK1/2 (Upstate, Charlottesville, VA); anti-phospho-ERK1/2 (Cell Signaling Technology, Danvers, MA); and anti-G protein-coupled receptor kinase 2 (GRK2) (Santa Cruz Biotechnology). In all studies, Abs were used at saturating dilutions unless otherwise indicated.
Anti cd44
Manufactured by Merck Group
Sourced in United States, United Kingdom
Anti-CD44 is a laboratory reagent used in cell biology research. It functions as an antibody that specifically binds to the CD44 protein, which is a cell surface receptor involved in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Anti sox2, by Abcam (2 mentions)
Anti cd133, by Novus Biologicals (2 mentions)
Anti tnc, by Abcam (2 mentions)
Anti lsd1, by Merck Group (2 mentions)
Anti sox9, by Abnova (2 mentions)
Anti gli1, by Abcam (2 mentions)
Anti 3 nitrotyrosine, by Santa Cruz Biotechnology (1 mentions)
Anti g protein coupled receptor kinase 2 grk2, by Santa Cruz Biotechnology (1 mentions)
Anti phospho erk1 2, by Cell Signaling Technology (1 mentions)
Cy5 conjugated anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Anti cd81, by BioLegend (1 mentions)
Anti cd63, by BioLegend (1 mentions)
Anti adam10, by Santa Cruz Biotechnology (1 mentions)
Tritc conjugated anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Anti acetylated α tubulin, by Santa Cruz Biotechnology (1 mentions)
Anti α tubulin, by Cell Signaling Technology (1 mentions)
Enhanced chemiluminescence kit, by Cytiva (1 mentions)
Anti dnmt1, by Merck Group (1 mentions)
Anti cd133, by Merck Group (1 mentions)
8 protocols using anti cd44
1
Antibody Characterization in Hematology
2
Immunostaining of Extracellular Vesicle Markers
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The following primary Abs were employed throughout this study: anti-CD9 clone P1/33/2 (Santa Cruz, Dallas, TX), anti-CD9 clone MEM-61 (Abcam, San Francisco, CA), anti-CD9 clone M-L13 (BD Biosciences, San Jose, CA), anti-CD9 clone HI9a (BioLegend, San Diego, CA), anti-CD81 (cat. #349502, BioLegend), anti-CD63 (cat. #sc-15363, Santa Cruz Biotechnology, Dallas, TX), anti-ADAM-10 (cat. #sc-25578, Santa Cruz Biotechnology), anti-β-actin (cat. #sc-47778, Santa Cruz Biotechnology), anti-IgSF8 (cat. #103561, Santa Cruz Biotechnology), anti-β1 integrin (cat #4706S, Cell Signaling Technology, Danvers, MA), anti-α-tubulin (cat. #sc-8035, Santa Cruz Biotechnology), anti-acetylated-α-tubulin (cat. #T7451, Sigma), anti-CD44 (cat. #MA5-13890, ThermoScientific, Waltham, MA). The following secondary Abs were employed: TRITC-conjugated anti-rabbit IgG (Jackson ImmunoResearch), TRITC-conjugated anti-goat IgG (Sigma), and Cy5-conjugated anti-mouse IgG (Jackson ImmunoResearch). For inhibition experiments, sodium azide was removed by desalting through Sephadex G-25 spin columns.
3
Protein Expression Analysis in U2OS and OSLC Cells
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The lysates were prepared by RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) from U2OS cells (1 × 106) or OSLCs (1 × 106). The protein concentrations were determined by the Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA). Electrophoresis with sodium dodecyl sulfate-polyacrylamide gel (10%, SDS-PAGE) was used to separate the lysate (40 μg protein), which was then transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore). The membranes were then blocked with TBST containing 5% BSA for 2 h at room temperature and then incubated with primary antibodies anti-β-actin (1 : 5000; catalog no. A5441; Sigma-Aldrich), anti-CD133, anti-CD44, anti-ABCG2, anti-DNMT1, anti-Bmi1, anti-Sox2, and anti-OCT4 (1 : 2000; catalog nos. 5860S, 3570S, 4477S, 3598S, 5856, 2748, and 2840 CST) overnight at 4°C. The membranes were then incubated with appropriate HRP-conjugated secondary antibodies (Beyotime Institute) for 1 h. The protein bands were visualized using an enhanced chemiluminescence kit (Amersham Biosciences) by using an enhanced chemiluminescence detection system (Ranon GIS-2008, Tanon Science & Technology Co., Ltd., Shanghai, China).
4
Immunohistochemical Staining of Paraffin Sections
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IHC and cell blocks were prepared as described earlier53 ,54 . Staining was performed on 2 µm-thick paraffin sections, which were incubated with Target Retrieval Solution (EnVision Flex, Dako, Carpinteria, California, USA) at pH 9 and then with primary antibodies (anti-FGFR1 (Abcam, Berlin, Germany), dilution 1:5000, anti-pAKT (Abcam), 1:100, or anti-CD44 (Sigma–Aldrich, Taufkirchen, Germany), 1:1000, at room temperature for 20 min. Secondary antibody was visualized by using DAB substrate, and contrasting was achieved by hematoxylin staining.
5
Characterization of GMSCs via Immunofluorescence
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GMSCs characterization using immunofluorescence was conducted based on the findings contained in the study authored by Jin et al.[18 (link)] Trypsin was added and centrifuged at 1600 rpm for 5 min. The pellets were added to 1 ml of α-Modified Eagle Medium (α-MEM) growth medium (Sigma Aldrich®, USA). GMSCs were cultured in a chamber slide (BD Biosciences, USA) suspended and grown in a 20-μl special glass. The glass object was placed in a box containing wet paper, incubated at 37°C for 1 h before, and 10% paraformaldehyde (Sigma-Aldrich Co.) was used to fix the cells. The result was then permeabilized in PBS and 1% Triton X100 for 3 min. GMSCs were stained with anti-CD34, anti-CD44, anti-CD45, anti-CD73, anti-CD90, and anti CD105 monoclonal antibodies (Sigma Aldrich®, USA) being added to each sample and then incubated at 37°C for 45 min. The PBS washing and drying process was repeated. At the next stage, an immunofluorescence examination of the glass object was conducted with 50% glycerin being added and the results observed through a fluorescent microscope (Automated Fluorescence Microscope, BX63, Olympus®, USA).
6
Western Blot Analysis of Stem Cell Markers
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Western blot analysis were carried out according to the standard procedures [8 (link)] for anti-TNC (1:1000, Abcam, UK), anti-LSD1 (1:2000, Sigma, USA), anti-SOX2 (1:1000, Abcam, UK), anti-CD44 (1:1000, Millipore, USA), anti-CD133 (1:1000, NOVUS, USA), anti-SOX9 (1:1000, Abnova, USA), anti-GLI1 (1:1000, Abcam, UK), and anti-β-actin (1:500, Bioss, China).
7
Anti-human Antibodies for Cell Profiling
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The anti-human antibodies that were used in the present study include: Anti-PTPRZ1 (cat no. 55125) from Sigma-Aldrich, St. Louis, MO, USA, and anti-pleiotrophin (cat no. 10821), anti-CD24 (cat no. 18330), and anti-CD44 (cat no. 60224) from Millipore, Billerica, MA, USA.
8
Immunohistochemical Profiling of CRC Samples
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IHC staining and evaluation of the IHC analysis on CRC tissue microarray samples was performed as previously described [8 (link)]. The primary antibody include anti-TNC (1:100, Abcam, UK), anti-SOX2 (1:100, Abcam, UK), anti-CD44 (1:80, Millipore, USA), anti-CD133 (1:100, NOVUS, USA), anti-LSD1 (1:250, Sigma, USA), anti-SOX9 (1:100, Abnova, USA), anti-p21 (1:100, Millipore, USA), anti-cyclinD1 (1:100, Millipore, USA), anti-p27 (1:100, Millipore, USA), anti-CDK4 (1:100, Millipore, USA), anti-p16 (1:100, Millipore, USA), anti-SMO (1:250, Santa, USA), and anti-GLI1 (1:100, Abcam, UK).
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