Anti-mouse galectin-3/Mac2 (galectin-3) [Rat IgG, 14–5301] was purchased from eBioscience Co.,Ltd. (San Diego, USA). Anti-Iba1 antibody [rabbit IgG, 019–19741] was purchased from Wako Pure Chemical (Osaka, Japan). The paraffinized sections were blocked to endogenous peroxidase activity by incubation in distilled water containing 3% hydrogen peroxide for 5 min. Antigen retrieval was performed, using a 0.01 M citrate buffer (pH 6.0) for both anti-galectin-3 and anti-Iba1 antibodies by the Pascal heat-induced target retrieval system (DAKO). Non-specific binding sites were blocked in 0.01 M phosphate-buffered saline (PBS), pH 7.4 containing 2% bovine serum albumin (BSA; Wako Pure Chemical, Osaka, Japan) for 30 min. Anti-galectin-3 and anti-Iba1 antibodies used at a dilution of 1:100, 1:500, respectively, in 2% BSA/PBS were added to the slides and incubated overnight at 4°C. Galectin-3 and Iba1 were detected with biotinylated anti-rat IgG (1:200, DAKO E0468), biotinylated anti-rabbit IgG (1:250, KPL-16-15-06) for 60 min, respectively, followed by incubation with avidin-coupled peroxidase (Vectastain ABC kit, Vector Laboratories) for 30 min. The peroxidase binding sites were detected by staining with 3,3’-diaminobenzidine (DAB) in 50 mM Tris-EDTA buffer. Finally, counterstaining was performed using Mayer’s hematoxylin.
Bovine serum albumin bsa
Manufactured by Fujifilm
Sourced in Japan, United States
Bovine serum albumin (BSA) is a common laboratory reagent used in various applications. It is a protein derived from bovine serum and serves as a stabilizing agent, blocking agent, and protein source in various biochemical procedures.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Merck Group (3 mentions)
Hydrochloric acid (hcl), by Fujifilm (3 mentions)
Nunc maxisorp plate, by Thermo Fisher Scientific (3 mentions)
Vectastain abc kit, by Vector Laboratories (2 mentions)
Entecavir, by Santa Cruz Biotechnology (2 mentions)
Bovine serum albumin (bsa), by Nacalai Tesque (2 mentions)
Trypsin, by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Fujifilm (2 mentions)
Tween 20, by Fujifilm (2 mentions)
Triton x 100, by Fujifilm (2 mentions)
Paraformaldehyde (pfa), by Nacalai Tesque (2 mentions)
Na2hpo4, by Fujifilm (2 mentions)
Sodium hydroxide (naoh), by Fujifilm (2 mentions)
Paraformaldehyde (pfa), by Fujifilm (2 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by Boehringer Ingelheim (2 mentions)
Glucose, by Fujifilm (2 mentions)
Sucrose, by Fujifilm (2 mentions)
Triton x 100, by Nacalai Tesque (2 mentions)
Dexamethasone (dex), by Fujifilm (2 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (2 mentions)
62 protocols using bovine serum albumin bsa
1
Immunohistochemical Analysis of Galectin-3 and Iba1
2
Screening of Compounds for Inhibiting HBV
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All the compounds used for screening were supplied from the RIKEN Natural Products Depository, NPDepo18 (link),19 (link). The purity and structures of NPD8716 and its derivatives were confirmed by LC-MS analysis. A preS1 peptide, consisting of 2–48 aa of the HBV preS1 region, and TAMRA-labeled preS1 peptide were synthesized (CS bio and Scrum, Inc.). Entecavir was purchased from Santa Cruz Biotechnology. Bovine serum albumin (BSA) was obtained from Wako.
3
VEGF-induced Cell Proliferation Assay
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Recombinant human VEGF, thiazolyl blue tetrazolium bromide (MTT), 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT), phenylmethylsulfonyl fluoride (PMSF), and 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) were purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA). RIPA buffer and chemiluminescent substrate were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA). Dimethyl sulfoxide, sodium fluoride (NaF), and bovine serum albumin (BSA) were from Wako Pure Chemical Industries (Osaka, Japan). Bradford reagent was purchased from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). MRBH, also called MGN-3 or BioBran, was provided by Daiwa Pharmaceutical Co., Ltd. (Tokyo, Japan).
4
DMSO-Based Compound Screening Protocol
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Dimethyl sulfoxide (DMSO), tetracycline, and bafilomycin A1 were purchased from Sigma-Aldrich. TAMRA-labeled preS1 peptide (preS1-TAMRA) was synthesized by CS bio. Cyclosporin A (CsA) was obtained from Enzo Lifesciences. Proanthocyanidin was purchased from Selleck. Entecavir was obtained from Santa Cruz. Bovine serum albumin (BSA) was purchased from FUJIFILM Wako Pure Chemical.
5
Lipid Synthesis and Secretion in iHep Cells
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To induce lipid synthesis, iHep cells were cultured in our hepato-medium (Sekiya and Suzuki, 2011 (link)) containing 20 ng/ml hepatocyte growth factor (HGF) (Sigma-Aldrich, St. Louis, MO, USA), 20 ng/ml epithelial growth factor (EGF) (Sigma-Aldrich), and 5% bovine serum albumin (BSA) (Wako) for 3 days with or without 1 mM oleic acid (Sigma-Aldrich), 1 mM elaidic acid (Tokyo Chemical Industry, Tokyo, Japan), or 1 mM palmitic acid (Wako). Hepatocytes isolated from adult mice by two-step collagenase digestion (Seglen, 1979 (link)) were also cultured under the same conditions used for iHep cells, except that HGF and EGF were omitted. MDFs were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 2 mM L-glutamine (Nacalai Tesque), penicillin/streptomycin (Nacalai Tesque), and 5% BSA for 3 days with or without 1 mM oleic acid. To induce lipid secretion, iHep cells were cultured in phenol red-free hepato-medium containing 20 ng/ml HGF, 20 ng/ml EGF, and 5% BSA for 48 h with or without 1 mM oleic acid. The culture medium was then changed to remove the oleic acid, and the supernatants were collected at 12, 24, and 36 h after changing the culture medium.
6
Measuring Cell Invasion Potential
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The invasiveness of cells was measured using Chemotaxicell filters (Kurabo Industries, Osaka, Japan) coated with Matrigel (Corning, NY, USA). Twenty-four hours after irradiation, cells were suspended in serum-free medium containing 0.1% bovine serum albumin (BSA) (Wako). Cells were added to the upper well (2 × 105 cells/well), and medium containing 10% FBS (chemoattractant) was added to the lower well. Cells were incubated for 24 h, fixed with 10%-buffered formalin, and then stained with hematoxylin and eosin (H&E). The number of cells on the lower side of the membrane was counted using a fluorescence microscope (×200 magnification).
7
Reagents and Materials for Cell Assays
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RPMI1640 medium, trypsin, Triton X-100, Brij 35, TriReagent and hexadimethrine bromide were purchased from Sigma-Aldrich (St. Louis, MO, USA). Nonidet P-40 was from Nacalai Tesque (Kyoto, Japan). Bovine serum albumin (BSA), gelatin, dimethyl sulfoxide (DMSO), phorbol 12-myristate 13-acetate (PMA) and polyethylene glycol (PEG) 6000 were purchased from Wako Pure Chemical Industries (Osaka, Japan). Polyethylenimine “Max” was purchased from Polysciences Inc. (Warrington, PA, USA). Fetal calf serum (FCS) was obtained from Biosera (Boussens, France). ASF104 serum-free medium was supplied by Ajinomoto (Tokyo, Japan). Matrigel was purchased from BD Biosciences (San Diego, CA, USA). The PrimeScript RT Reagent Kit and KAPA SYBR FAST qPCR Kit Master Mix (2×) ABI Prism were obtained from Takara Bio Inc. (Shiga, Japan) and KAPA Biosystems (Boston, MA, USA), respectively. ViraPower Lentiviral Packaging Mix and puromycin dihydrochloride were supplied by Life Technologies (Carlsbad, CA, USA). Oligonucleotides were supplied by FASMAC (Kanagawa, Japan). Recombinant murine TNF-α was a product of Peprotech (Rocky Hill, NJ, USA). A fluorescent dye, 3′-O-acetyl-2′, 7′-bis(carboxyethyl)-4 or 5-carboxyfluorescein, diacetoxymethyl ester (BCECF-AM), was obtained from Dojindo Laboratories (Kumamoto, Japan). Gelatin Sepharose 4B was a product of GE Healthcare (Piscataway, NJ, USA).
8
Immunohistochemical Analysis of BDNF and SYP
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Anti-rat BDNF antibody [rabbit IgG ab182199] and anti-rat SYP antibody [rabbit IgG, ab8049] were purchased from Bay Bioscience (Hyogo, Japan) and Wako Pure Chemical (Osaka, Japan), respectively. The paraffinized sections were blocked from endogenous peroxidase activity by incubation in distilled water containing 3% hydrogen peroxide for 5 min. Antigen retrieval was performed, using a 0.01 M citrate buffer (pH 6.0) for both anti-rat BDNF antibody and anti-rat SYP antibodies by the Pascal heat-induced target retrieval system (DAKO Japan, Tokyo, Japan). Nonspecific binding sites were blocked in 0.01 M phosphate-buffered saline (PBS), pH 7.4, containing 2% bovine serum albumin (BSA; Wako Pure Chemical) for 60 min. Both anti-rat BDNF and anti-rat SYP antibodies used at a dilution of 1 : 100 in 2% BSA. PBS were added on the slides and incubated overnight at 4°C. BDNF and SYP were detected with a biotinylated anti-rabbit IgG (1 : 1000) for 30 min, followed by incubation with avidin-coupled peroxidase (Vectastain ABC Kit, Vector Laboratories, Burlingame, USA) for 30 min.
9
Immunofluorescent Detection of OGA Expression
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Expression of OGA was detected by immunofluorescent staining in diploids from the 2-cell to blastocyst stage. Diploids were collected as described for detection of RNA expression. Subsequently, diploids were fixed and permeabilized with PBS-PVA that contained 0.2% (v/v) Triton X-100 (Sigma-Aldrich) and 4% (w/v) paraformaldehyde (PFA, Sigma-Aldrich) for 25 min and washed twice in PBS-PVA. Fixed diploids were stored at 4 C in PBS-PVA containing 1% (w/v) bovine serum albumin (BSA; Wako) (BPP) before use. Immunofluorescence microscopy was carried out as described below. Diploids were treated with rabbit anti-human OGA antibody (1:100; Proteintech Group, Chicago, IL, USA) in BPP at 4 C overnight. Then they were washed three times in PBS-PVA and treated with Alexa Fluor 568 anti-rabbit IgG antibody (Life Technologies) in BPP for 1 h at room temperature. After washing in PBS-PVA, diploids were mounted in ProLong Gold Antifade Mountant (Life Technologies) containing
4',6-diamidino-2-phenylindole (DAPI) and observed under an epifluorescence microscope (BX53-FL; Olympus, Tokyo, Japan).
4',6-diamidino-2-phenylindole (DAPI) and observed under an epifluorescence microscope (BX53-FL; Olympus, Tokyo, Japan).
10
Extracellular DNA Quantification Protocol
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Extracellular DNA was stained with 5 μm SYTOX Green (Life Technologies, Carlsbad, CA, USA), a fluorescent membrane‐impermeable DNA dye. Fluorescence was quantified using a microplate reader equipped with filters to detect excitation/emission maxima of 504/523 nm (EnSpire; PerkinElmer, Waltham, MA, USA). When indicated, neutrophils were stimulated with 20 or 200 nm PMA, or cultured in the presence of 100 U·mL−1 DNase I (TaKaRa, Osaka, Japan), 40 mg·mL−1 bovine serum albumin (BSA; Wako Pure Chemical Industries, Osaka, Japan), 40 mg·mL−1 human serum albumin (Wako), heat‐treated (complement‐inactivated) serum, protein G/A sepharose‐treated (immunoglobulin‐depleted) serum, 10 mm EDTA, or 5 or 20 mm N‐acetyl‐l ‐cysteine (NAC; Wako).
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