Fluorescence-activated cell sorting (FACS) was used to characterize canine Ad-MSCs from passage 2. Cultured cells were trypsinized, spun down in a centrifuge, and washed twice in FACS buffer consisting of 10 mM hepes (Gibco/Invitrogen), 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mg/mL bovine serum albumin (BSA) (Sigma-Aldrich) in Leibovitz's L-15 medium (Gibco/Invitrogen). After the washing step, cells aliquots (1 × 106 cells) were incubated in FACS buffer containing fluorochrome-conjugated monoclonal antibodies against CD29, CD90 and STRO-1 (R&D Systems, Minneapolis, MN, USA), CD34 and CD45 (MiltenyiBiotech SL), and MHC-II (BD Pharmingen, Becton Dickinson), or an appropriate isotype control antibody (Sigma-Aldrich). After 30 minutes in the dark on ice, cells were washed again in FACS buffer before analysis. Five hundred thousand events per sample were analyzed on aMoFlo SP1338 (DakoCytomation, Denmark) using Summit software. Cells were gated on forward and side scatter to exclude debris and cell aggregates, and dead cells were excluded by 7-aminoactinomycin D (7-AAD, BD Pharmingen) staining.
Isotype control antibody
Manufactured by Merck Group
Sourced in United States
Isotype control antibodies are laboratory reagents used to establish appropriate background signal levels in immunoassays. They are antibodies that have the same isotype as the primary antibody being used but do not bind to the target antigen. Isotype controls help identify non-specific binding and set appropriate gating parameters in flow cytometry and other applications.
Automatically generated - may contain errors
Lab products found in correlation
3 3 diaminobenzidine tetrahydrochloride dab, by Vector Laboratories (2 mentions)
7 aminoactinomycin d 7 aad, by BD (1 mentions)
Leibovitz s l 15 medium, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Peroxidase conjugated streptavidin, by Vector Laboratories (1 mentions)
Biotinylated anti mouse igg, by Vector Laboratories (1 mentions)
Opti mem medium, by Thermo Fisher Scientific (1 mentions)
Cm5 sensor chip, by GE Healthcare (1 mentions)
T4 dna ligase, by Takara Bio (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
E coli dh5α, by Takara Bio (1 mentions)
Co ip kit, by Thermo Fisher Scientific (1 mentions)
Immobilon p transfer membrane, by Merck Group (1 mentions)
Qiaprep spin miniprep kit, by Qiagen (1 mentions)
Amine coupling kit, by GE Healthcare (1 mentions)
Mouse monoclonal anti ha antibody, by Merck Group (1 mentions)
3 30 diaminobenzidine tetrahydrochloride dab, by Merck Group (1 mentions)
Anti p53 antibody fl393, by Santa Cruz Biotechnology (1 mentions)
Bortezomib, by Enzo Life Sciences (1 mentions)
10 protocols using isotype control antibody
1
Canine Adipose-Derived Mesenchymal Stem Cell Characterization
2
Modulating Colon Cancer Progression
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BALB/c mice were given 500 μg α-IL-25 (Amgen) via i.p. injection on days 7, 10, 14, 17, 21, 28, 35, 42, 49, 56, and 63 of colitis induced colon cancer model. Control animals were given an equivalent dose of isotype control antibody (Sigma-Aldrich). Anti-IL-25 was a gift from Amgen (Thousand Oaks, CA).
3
Immunohistochemical Analysis of EMT Markers
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5-μm sections of paraffin-embedded tissues were treated with pepsin or 30 min and blocked with BSA for 30 min at room temperature. Tissue sections were then incubated with antibodies to DAL-1, E-cadherin, Snail or vimentin overnight at 4°C. Isotype control antibody (Sigma-Aldrich) was used as a negative control. Each slide was washed three times in TBS and incubated with biotinylated anti–mouse IgG (Vector Laboratories) in a humid chamber for 30 min. The positive cells were detected using peroxidase-conjugated streptavidin (Vector Laboratories) followed by 3′3-diaminobenzidine tetrahydrochloride (DAB; Vector Laboratories). The slides were counterstained with hematoxylin.
4
Eukaryotic Expression Plasmid Construction
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The recombinant plasmids pET30a-H (GenBank Accession No. X74443), pCAGGS-F (GenBank Accession No. X74443) and vector pCMV-HA were provided by the Lanzhou Veterinary Research Institute of Chinese Academy of Agricultural Sciences and were used to construct eukaryotic expressing plasmids. CHO-K1 cells were obtained from Shanghai Institutes for Biological Sciences (SIBS). E. coli DH5α, T4 DNA ligase and all restriction enzymes were purchased from TaKaRa. The QIAprep® spin miniprep kit was from QIAGENE. F12 K, G418, OPTI-MEM medium and Lipofectamine3000 were products of Invitrogen. FCS was purchased from Gibco BRL Life Tech. Co-IP kit was purchased from Thermo Fisher. Mouse anti-HA monoclonal antibody, isotype control antibody, lexa Fluor 488/HRP-conjugated anti-mouse IgG and 3,30-diaminobenzidine tetrahydrochloride (DAB) were purchased from Sigma. Immobilon-P transfer membranes were purchased from Millipore. CM5 sensor chip, amine coupling kit and all solutions were also purchased from GE Healthcare.
5
Immunoprecipitation of p53 Protein
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For immunoprecipitation, cells were treated for 4 h with Etoposide and/or Roc-A in the absence or presence of 100 nM Bortezomib (Enzo Life Sciences). In the case of metabolic pulse-labeling experiments, treatment was followed by adding 100 μCi/ml of [35S]-methionine-labeling mix (PerkinElmer, Waltham, MA, USA) to the medium for 0–15 min. Subsequently, cells were washed in ice-cold PBS, lysed in IP buffer (20 mM Tris-HCl, 5 M NaCl, 2 mM EDTA, 1% Triton X-100, protease inhibitors) and centrifuged (10 000 × g, 20 min) to clear lysates. Aliquots were taken for input control and lysates were incubated overnight with sepharose-coupled protein A beads, anti-p53 antibody (FL-393; Santa Cruz) or isotype control antibody (Sigma-Aldrich). Two wash steps with IP buffer preceded boiling of beads in denaturing sample buffer at 95°C for 5 min. Incorporation of [35S]-methionine into p53 protein was detected by the phosphoimaging system FLA-7000 IR (Fujifilm Europe GmbH, Düsseldorf, Germany).
6
Cell Surface Localization of HATL5
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Cell-surface imaging was performed using HEK293 or COS-7 cells transfected with human full-length HATL5-V5. Cells were seeded on coverslips coated with rat type-2 collagen (BD Biosciences, Franklin Lakes, NJ) and allowed to adhere and grow for 36 h, at which point, media was removed and cells were fixed in 4% paraformaldehyde in PBS for 30 min at room temperature. HATL5-V5 was detected using a monoclonal anti-V5 antibody (Invitrogen, Life Technologies, Grand Island, NY) or an isotype control antibody (Sigma, St. Louis, MO) and a secondary AlexaFlour-568-conjugated goat-anti-mouse antibody (Invitrogen, Life Technologies, Grand Island, NY). Cell staining was imaged by confocal microscopy using a Leica TCS SP2 confocal microscope (Leica, Buffalo Grove, IL).
7
IL-16 Neutralizing Antibody Cytotoxicity Assay
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Cells were washed twice with PBS, seeded in duplicates at 2×105 cells/ml in a 96 well plate and treated with increasing concentrations of an IL-16 neutralizing antibody (clone 14.1, EMD Millipore, Darmstadt, Germany) or an isotype control antibody (Millipore). After 12 h cells were harvested and analyzed on a beta scintillation counter.
8
Immunoprecipitation of p53 in HCMV-infected HMECs
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HMECs were mock-treated or were infected with HCMV strains (MOI = 1). At day 3 post infection, the cell lysates were precleared by adding 50 μl of protein A magnetic beads (Millipore) for 1 h at 4 °C. The clear supernatants were removed, combined with anti-p53 antibody (Cell Signaling) or isotype control antibody (Millipore) and incubated overnight at 4 °C. The lysates were further incubated with 50 μl protein A magnetic beads (Millipore) at 4 °C for 2 h. Immune complex were washed in the presence of protease inhibitors (Roche, Meylan, France) and bound protein was eluted with sample buffer and run on 10% SDS-PAGE gels. Expression of IE2 was determined by western blotting.
9
Immunohistochemical Analysis of AIRE Expression
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Deparaffinized sections of the thymus were washed, and endogenous peroxidase activity was quenched by immersion in 3% hydrogen peroxide in methanol. Sections were then incubated with antiautoimmune regulator (AIRE) antibodies (1 : 200 dilution, Sigma-Aldrich, St. Louis, MO, USA) for 1 h. Isotype control antibodies (Sigma-Aldrich) were used under the same conditions. A SuperPicTure Polymer Detection Kit (Zymed/Invitrogen, Carlsbad, CA, USA) was used for enzymatic immunohistochemical staining in accordance with the manufacturer's protocols.
10
Histological Analysis of Adipose, Liver, and Pancreas
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Mice were sacrificed at 16 weeks, following the start of DIO. Mouse EWAT, liver, and pancreas were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) (Sigma Aldrich, St. Louis, MO) for routine histological analysis. For immunohistochemistry (IHC), 5 μm sections of paraffin-embedded EWAT were treated with Proteinase K for 30 min, and blocked with 5% BSA and 0.5% Tween 20 for another 30 min at room temperature. To examine the degree of macrophage infiltration, EWAT, liver, and pancreas sections were incubated with macrophage marker antibody (RM0029-11H3; Santa Cruz Biotechnology, CA) at room temperature for 1 h. Isotype control antibodies (Sigma Aldrich, St. Louis, MO) were used as a negative control. Each slide was washed three times in phosphate buffered saline with Tween 20 (PBST). After the final wash step, the staining was visualized using the EnVision TM Detection System (Dako, Glostrup, Denmark) in a humid chamber for 30 min. Positive cells were detected using 3,3'diaminobenzidine tetrahydrochloride (DAB; Vector Laboratories, San Francisco, CA).
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