The bovine serum albumin (BSA)-stabilized IL-4 MDs was fabricated according to previous studies.18 (link),19 (link),100 ng/mL IL-4 (R&D system, China) was used based on a previous study.20 (link) Briefly, 100 ng/mL IL-4, 300 μL perfluorocarbon, and 4 mL of PBS, and 40 mg of bovine serum albumin (BSA, Sigma Aldrich, China) were used for MDs fabrication. The resulting emulsion was ultracentrifuged (Beckman Coulter, Optima XPN-100, USA) at 14,000 rpm for 30 min, and the MDs-IL4 were used for subsequent testing. To generate fluorescence-labeled MDs for fluorescence imaging, fluorescein isothiocyanate (FITC)-labelled BSA (Sigma Aldrich, China) was used for MDs fabrication. Fluorescence-labeled MDs-IL4 were used for MDs characterization using a confocal laser scanning microscopy (CLSM). Briefly, 50 mg MDs-IL4 were diluted into PBS and then observed using a confocal laser scanning microscope with a ×40 objective (Leica DM IRB; Leica, Wetzlar, Germany). MDs-IL4 growth and rupture were observed using an inverted light microscope (Leica, Wetzlar, Germany). Briefly, the samples were exposed to an ultrasound probe with an acoustic frequency 1 MHz by portable home use ultrasound pain therapy device for 25 min (MYCHWAY, China). Images were captured using an inverted light microscope (Leica, Wetzlar, Germany).
Bovine serum albumin (bsa)
Manufactured by R&D Systems
Sourced in United States, United Kingdom
BSA (Bovine Serum Albumin) is a widely used protein commonly used in laboratory applications. It serves as a stabilizing agent, blocking agent, and protein standard. BSA is a purified protein derived from bovine serum.
Automatically generated - may contain errors
Lab products found in correlation
Bovine serum albumin (bsa), by Merck Group (12 mentions)
Triton x 100, by Merck Group (7 mentions)
L glutamine, by Thermo Fisher Scientific (3 mentions)
N2 supplement, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Fibroblast growth factor 2 (fgf2), by R&D Systems (2 mentions)
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (2 mentions)
Dexamethasone (dex), by Merck Group (2 mentions)
Tgf β1, by R&D Systems (2 mentions)
Paraformaldehyde (pfa), by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (2 mentions)
Tcs sp8, by Leica (2 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Luminata forte western hrp substrate, by Merck Group (2 mentions)
Hcl pbs, by R&D Systems (2 mentions)
Fluoromount g containing dapi, by Thermo Fisher Scientific (2 mentions)
Sp8 confocal microscope, by Leica (2 mentions)
Antigenfix, by Microtech (2 mentions)
Egm 2 bulletkit, by Lonza (2 mentions)
67 protocols using bovine serum albumin (bsa)
1
Fabrication and Characterization of Ultrasound-Responsive IL-4 Microdroplets
2
Quantification of Mouse and Human Cystatin E/M
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Recombinant mouse Cst6 and human CST6 (both from R&D Systems, Minneapolis, MN, USA) were used as a standard and as a negative control, respectively, in concentrations varying from 5 to 0.156 ng/ml. The wells of a 96-well plate were coated overnight at 4°C with polyclonal rabbit anti-human CST6 antibody (2 (link)), followed by incubation with 1% bovine serum albumin (ICN Biomedicals, Aurora, OH, USA) and 1% normal goat serum (Vector Laboratories, Burlingame, CA, USA) in PBS for 30 min. Subsequently, standards, controls, and samples (undiluted up to 32× diluted) were incubated for 1 h, followed by incubation with monoclonal rat anti-mouse Cst6 (R&D Systems) in PBS/1% normal rabbit serum/0.1% bovine serum albumin/0.05% Tween-20 for 30 min. Next, wells were incubated with goat anti-rat biotinylated antibody (Vector Laboratories) for 30 min, followed by a final incubation with avidin-biotinylated horseradish peroxidase complex (Vector Laboratories) for 30 min. The above incubation steps were performed at 37°C and separated by repeated washing steps with PBS/0.05% Tween-20. Chromogenic substrate 1-step Ultra TMB (Thermo Fisher Scientific) was used as substrate for detection and the reaction was stopped by adding 4 M H2SO4. Each well was measured for mouse Cst6 at an absorbance of 450 nm with an ELISA microplate reader (Bio-Rad, Hercules, CA, USA).
3
Immunohistochemical Analysis of Tumor Xenografts
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Tumor xenografts were fixed in 4% neutral formalin at room temperature for 48 h, soaked in 4% paraffin, and were cut into 4-µm-thick sections. Xylene was applied for deparaffinising. Next, rehydration was implemented with an ethanol gradient. After culture with 0.3% H2O2 for 30 min and blocking with 5% bovine serum albumin (R&D Systems) for 45 min at 37°C, the slides were incubated all night with E2F6 (product code ab53061; 1:1,000 dilution) or Ki-67 (product code ab15580; 1:1,000 dilution; both from Abcam) at 4°C, followed by 45 min of treatment at room temperature with a horseradish peroxidase-conjugated secondary antibody (cat. no. ab205718; Abcam; 1:500 dilution). Then, 3,3′-diaminobenzidine (DAB) color reagent was applied to detect the antibody binding, and tumor xenografts were counterstained with 1% hematoxylin at room temperature for 10 min and dehydrated in ethanol. Finally, image acquisition was implemented utilizing a light microscope (×200, magnification).
4
Quantifying HDAC4 and Ki-67 Expression in Tumor Xenografts
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HDAC4 and Ki-67expression levels in tumor xenografts were examined via IHC. Tumor xenografts were fixed in 4% neutral formalin and soaked in 4% paraffin, after which the xenografts were cut into 4-μm-thick sections. Deparaffinizing was achieved using xylene, followed by rehydration with an ethanol gradient. Following incubation with 0.3% H2O2 for 30 min and blocking with 5% bovine serum albumin (R&D Systems) for 45 min at 37 °C, the slides were treated with HDAC4 (cat. no. ab235583) or Ki-67 (cat. no. ab15580; all from Abcam) at 4 °C overnight. Thereafter, a horseradish peroxidase-conjugated secondary antibody (cat. no. ab205718; Abcam; 1:500 dilution) was applied to incubate the slides at room temperature for 45 min. Subsequently, 3,3′-diaminobenzidine (DAB) color reagent was added to detect the antibody binding, and tumor xenografts were counterstained with 1% hematoxylin at room temperature for 3 min and dehydrated in ethanol. Image acquisition was conducted using an Olympus microscope.
5
Immunohistochemical Analysis of Tumor Xenografts
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The fixation of tumor xenografts in 4% neutral formalin was conducted at room temperature for 24 h, after which was then embedded in paraffin. After the paraffin is cut into 4 μm sections, they were deparaffinized in xylene, rehydrated in a graded alcohol series and probed with boiling citrate buffer. In order to decrease the non-specific binding, the sections were probed with 5% bovine serum albumin (R&D Systems, Minneapolis, MN, USA) at 37 °C for 45 min. After 16 h incubation with Ki-67 antibody (sc-23900; Santa Cruz Biotechnology, Dallas, TX, USA) at 4 °C, the sections were further incubated with a horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h. Thereafter, the sections were counterstained with 1% hematoxylin at room temperature for 5 min, followed by dehydration in a graded series of ethanol. Finally,the iamges were capatured under an light microscope.
6
BRAF-mutant Thyroid Cancer Cell Lines
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PTC cell lines (BCPAP and K1 were both harboring the BRAFV600E mutation) were grown in Roswell Park Memorial Institute 1640 (RPMI1640) or Dulbecco’s Modified Eagle Medium (DMEM)/F12 (1:1) medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), and both were supplemented with 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA) at 37°C with 5% CO2. Recombinant human TGF-β1 (7754-BH, R&D systems, Minneapolis, MN, USA) was reconstituted at 100 μg/mL in sterile 4 mM HCl containing at least 0.1% bovine serum albumin (BSA). LGZ was provided by Chong Kun Dang Pharm. Inc, and another conventional PPAR-γ ligand, RGZ (122320-73-4, Sigma-Aldrich, St. Louis, MO, USA), and PPAR-γ antagonist-2-chloro-5-nitrobenzanilide (GW9662) (M6191, Sigma-Aldrich) were purchased and dissolved in dimethyl sulfoxide (DMSO) and stored at −20°C. This study followed Institutional Review Board of Seoul St. Mary’s Hospital, The Catholic University of Korea, which indicated for exemption (Number: KC-20EISI0390).
7
Tumor Xenograft Immunohistochemical Staining
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Tumor xenografts were fixed in 4% neutral formalin at room temperature for 24 h, embedded in paraffin and cut into 4 μm sections. The sections were then deparaffinized in xylene, rehydrated in a graded alcohol series and treated with boiling 0.01 mol/l citrate buffer for 15 min for antigen retrieval. Endogenous peroxidase activity was blocked with hydrogen peroxide (0.3%) at room temperature for 15 min, and the sections were incubated with 5% bovine serum albumin (R&D Systems, Minneapolis, MN, USA) at 37 °C for 45 min to reduce non-specific binding. Immunostaining with MET antibody (#8198; Cell Signaling Technology) or Ki-67 (ab15580; Abcam, Cambridge, UK) were carried out at 4 °C for 16 h, followed by incubation with a horseradish peroxidase (HRP) -conjugated secondary antibody from the Envision kit (Dako; Agilent Technologies, Inc., Santa Clara, CA, USA) for 45 min at room temperature. Antibody binding was detected by DAB (Dako; Agilent Technologies, Inc.), according to manufacturer's protocol, at room temperature for 1 min and the reaction was terminated by immersion of tissue sections in distilled water once brown staining appeared. Tissue sections were counterstained with 1% hematoxylin at room temperature for 3 min and dehydrated in a graded series of ethanol. Images of representative fields were obtained from Nikon Digital ECLIPSE C1 microscope (Nikon Corporation).
8
Rh-E-selectin Adhesion Assay for Capan-1 Cells
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Rh-E-selectin (5 g/ml; R & D Systems, Inc., Minneapolis, MN, USA) or 1% bovine serum albumin (cat. no., A8020; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) was coated onto 96-well microplates at 4°C for 24 h. Following washing 3 times with assay buffer (20 mM Hepes, pH 7.4 with 150 mM NaCl and 1 mM CaCl2), 1×105 Capan-1:shNT (shRNA non-targeting) and Capan-1:shFUT3 cells were added. Followed by incubation at 37°C for 1 h, adherent cells were examined with 50 µl (5 mg/ml) Thiazolyl Blue (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) based on a colorimetric method. Plates were read using spectrophotometric analysis at a wavelength of 570 nm using the SpectraMax M5e microplate reader (Molecular Devices, LLC, Sunnyvale, CA, USA) and SoftMax Pro version 6.4 software (Molecular Devices). All the experiments were repeated in quintuplicate, and three independent assays were undertaken. Numbers of adhered cells are presented as mean ± standard deviation (SD).
9
Acetylshikonin Induces Apoptosis in HCC Cells
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Human hepatocellular carcinoma cells HepG2, Huh7 and normal hepatic cell LO2 were provided by Dr. Wu in The University of Kansas. Wild-type and p53−/−Hep3B cells, wild-type and p53 upregulated modulator of apoptosis (PUMA)−/−Hep3B cells, HepG2 Bax+/− and HepG2 Bax−/− Cells were provided by Prof. Shi in the University of Kansas. Cells were grown in Roswell Park Memorial Institute (RPMI) 1640 supplemented with penicillin (5 units/mL), streptomycin (5 ug/mL), and 10% heat-inactivated fetal bovine serum (FBS). Acetylshikonin was obtained from Huakang Pharmaceutical Company (Deyang, China) and resolved in 0.1% dimethyl sulfoxide (DMSO). zlETD-fmk, zlEHD-fmk, zDEVD-fmk and N-acetyl-l-cysteine (NAC) were purchased from Sigma-Aldrich Corp (St. Louis, MO, USA). Recombinant human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) was purchased from R&D Systems (Wiesbaden, Germany) and resolved in PBS + 0.01% bovine serum albumin (BSA).
10
Cell Viability and Migration Assays
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Cell viability was evaluated using an MTT assay (Sigma-Aldrich; Merck KGaA). Cells were digested with trypsin, and 10% FBS was added to prevent serum interference. The cell concentration was adjusted to 4×104 cells/well. A total of 20 µl MTT stock solution was added per well and incubated in 37°C for 4 h. The samples were centrifuged and dimethyl sulfoxide was then added. Following 10 min of incubation, the absorbance values were recorded at 490 nm and the growth curve was drawn.
For migration, a Transwell assay was performed with a Transwell chamber (ECM550, Chemicon International, Inc., Billerica, MA, USA) was used. In the upper chamber, 1×105 NSCs in 100 µl DMEM/F12 and 0.05% bovine serum albumin (R&D Systems, Inc., Minneapolis, MN, USA) were added. In the lower chamber, 300 µl DMEM/F12 with 10% FBS medium was added. Following 24 h incubation, the cells were stained with 1% crystal violet for 20 min at room temperature and counted under a light microscope (magnification, ×100).
For migration, a Transwell assay was performed with a Transwell chamber (ECM550, Chemicon International, Inc., Billerica, MA, USA) was used. In the upper chamber, 1×105 NSCs in 100 µl DMEM/F12 and 0.05% bovine serum albumin (R&D Systems, Inc., Minneapolis, MN, USA) were added. In the lower chamber, 300 µl DMEM/F12 with 10% FBS medium was added. Following 24 h incubation, the cells were stained with 1% crystal violet for 20 min at room temperature and counted under a light microscope (magnification, ×100).
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