Microdissection of formalin-fixed, paraffin-embedded tissue was performed on hematoxylin-stained slides for both tumor and non-neoplastic mucosae. The tumor and non-neoplastic mucosal components were microdissected separately. In brief, the tissue blocks were scratched at both lateral margin of tumor tissue for marking the surface of the object tissue. Subsequently, histological sections were removed from the histological block (up to 10 µm). We confirmed that the histological sections contained at least 50% tumor tissue. Microdissected tissue was incubated at 56 °C for 12–18 h in 50 µl buffer containing 0.5% Tween-20 (Boehringer Mannheim, Mannheim, Germany), 20 µg proteinase K (Boehringer Mannheim), 50 mmol/l Trizma base, pH 8.9, and 2 mmol/l ethylenediaminetetraacetic acid. proteinase K was inactivated by incubating the samples at 100 °C for 10 min.
Tween 20
Manufactured by Roche
Sourced in Germany
Tween-20 is a non-ionic detergent commonly used as a laboratory reagent. It is a polysorbate surfactant that helps solubilize and stabilize proteins and other biomolecules in aqueous solutions. Tween-20 is widely used in various biochemical applications, including Western blotting, ELISA, and protein purification.
Automatically generated - may contain errors
Lab products found in correlation
Proteinase k, by Roche (4 mentions)
Trizma base, by Roche (4 mentions)
Bovine serum albumin (bsa), by Roche (2 mentions)
Rbc lysis buffer, by Thermo Fisher Scientific (1 mentions)
Lymphoprep, by Axis-Shield (1 mentions)
Glycine, by Serva Electrophoresis (1 mentions)
3 4 hydroxyphenyl propionic acid hppa, by Serva Electrophoresis (1 mentions)
Iblot system, by Thermo Fisher Scientific (1 mentions)
Enhanced chemi luminescence (ecl), by Cytiva (1 mentions)
Tween 20, by Merck Group (1 mentions)
Mouse monoclonal anti gfp, by Roche (1 mentions)
Amine coupling kit, by GE Healthcare (1 mentions)
Caov3, by American Type Culture Collection (1 mentions)
Nci h226, by American Type Culture Collection (1 mentions)
293fectin, by Thermo Fisher Scientific (1 mentions)
Cell culture medium, by Thermo Fisher Scientific (1 mentions)
Pcdna3.1 vector, by Thermo Fisher Scientific (1 mentions)
Cm5 sensor chip, by GE Healthcare (1 mentions)
Human antibody capture kit, by GE Healthcare (1 mentions)
Nci h358, by American Type Culture Collection (1 mentions)
13 protocols using tween 20
1
Microdissection of FFPE Tissue
2
Tumor Microdissection and DNA Extraction
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Microdissection of formalin-fixed, paraffin-embedded tumor and non-tumor mucosal sections was performed on hematoxylin-stained slides. The tumor and non-tumor mucosal components were microdissected separately and incubated in 50 μL buffer (0.5% Tween-20 [Boehringer Mannheim, Ingelheim, Germany], 20 μg proteinase K [Boehringer Mannheim], 50 mM Trizma base, pH 8.9, and 2 mM ethylenediaminetetraacetic acid) at 56 °C for 12–18 h. proteinase K was inactivated by incubating the samples at 100 °C for 10 min. All tumor samples in which the neoplastic cells accounted for at least 50% of the cell population were evaluated.
3
Gastric Tumor Tissue Sampling and DNA/RNA Extraction
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Tumour samples were obtained from resected stomach tissues using biopsy forceps within 10–20 min of resection. Normal gastric mucosa distant from the tumour was removed from the mucosa using biopsy forceps; as a control (germline data), gastric biopsy samples from patients with IMN with chronic gastritis were included. Tumour tissues for pathological analysis were obtained from a region of the resected stomach adjacent to the site used for molecular analysis (one sample was obtained as a representative sample). In addition, the tumour tissue samples examined were surrounded by tumour tissue on all sides to ensure the presence of tumour tissue in the sample (Figure 1 ). In this section, the proportion of tumour cells accounted for at least 50% of the tissue. If other histological components were contained in the histological section, genomic DNA and RNA were extracted from whole tumour tissues. In brief, microdissected tissues were incubated at 56 °C for 12–18 h in 50 μL buffer containing 0.5% Tween‐20 (Boehringer Mannheim, Mannheim, Germany), 20 μg proteinase K (Boehringer Mannheim), 50 mm Trizma base (pH 8.9), and 2 mm ethylenediaminetetraacetic acid. proteinase K was inactivated by incubating the samples at 100 °C for 10 min. A representative example is shown in Figure 1 .
4
Microdissection and DNA Extraction from FFPE Tissue
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Microdissection of formalin-fixed, paraffin-embedded tissues was performed on hematoxylin-stained slides for three components, including head lesions, base lesions, and non-neoplastic mucosa. The three components were microdissected separately. Briefly, the tissue blocks were scratched at both the lateral margin of the tumor tissue to mark the surface of the target tissue. Subsequently, histological sections were removed from the histological block (up to 10 µm). We confirmed that the histological sections contained at least 50% tumor tissue. Microdissected tissue was incubated at 56 °C for 12–18 h in 50 µL buffer containing 0.5% Tween-20 (Boehringer Mannheim, Mannheim, Germany), 20 µg proteinase K (Boehringer Mannheim), 50 mM Trizma base (pH 8.9), and 2 mM ethylenediaminetetraacetic acid. proteinase K was inactivated by incubating the samples at 100 °C for 10 min.
5
Pharmacokinetics of ATV-RTV-TFV DcNP
6
Reagents for Immunoassay Development
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Bovine serum albumin (BSA), Roche universal buffer for ELISA (RUB), phosphate-buffered saline (PBS), and Tween20 (10% (w/v)) were obtained from Roche Diagnostics GmbH (Mannhein, Germany). Rat serum (female, Wistar) was from Charles River Laboratories (Sulzfeld, Germany). Ethanol, 30% H2O2, and Tris(hydroxymethyl)aminomethane (Tris/HCl) were ordered from Merck KGaA (Darmstadt, Germany). Glycine and 3-(4-Hydroxyphenyl) propionic acid (HPPA) were obtained from Serva Electrophoresis GmbH (Heidelberg, Germany) and Sigma Aldrich (St. Louis, MO, USA), respectively.
7
Immunoblot Analysis of Malaria Parasite Proteins
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For immunoblot analysis, tightly synchronised parasites were harvested by centrifugation at the appropriate time, released from erythrocytes in 1.5 volumes of 0.15% saponin+cOmplete protease inhibitor (Roche Applied Biosciences) for 10 min on ice, washed with ice-cold PBS and solubilised in 2× SDS loading buffer. Proteins were separated on NuPAGE Novex 4–12% Bis-Tris gels with MES Buffer (Life Technologies) and transferred to 0.2 μm PVDF membrane using the iBlot system (Life Technologies). Membranes were blocked in PBS containing 5% (w/v) skimmed milk and 0.1% Tween 20 (Sigma-Aldrich) for 1 h at room temperature then incubated for 1 h with primary antisera diluted as follows in PBS containing 1% (w/v) skimmed milk and 0.1% Tween 20: affinity-purified rabbit anti-MTRAP TSR (rM-tsr), 1:500 (Baum et al., 2006b (link)); rabbit anti-MTRAP mid monoclonal (rM-mid), 1:25 (this study); rabbit anti-MTRAP tail (rM-tail), 1:1000 (this study); mouse anti-GFP monoclonal, 1:1000 (clones 7.1/13.1, Roche Applied Biosciences); and anti-MSP1 (Cooper et al., 1992 (link)), 1:1000. After washing, signal was detected with appropriate IgG horseradish peroxidase (HRP) conjugates (Merck Millipore), followed by further washes and chemiluminescence detection (ECL, Amersham Biosciences).
8
Ovarian Cancer Cell Sensitivity Screening
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NCI-H226, NCI-H358 and NIH:OVCAR3 cells were obtained from ATCC. The ovarian cancer cell lines screened for RG7787 sensitivity at Horizon Inc. were obtained from the following providers: ATCC (Caov-3, Caov-4, and OVCAR3), Public Health England’s European Collection of Authenticated Cell Cultures (59M, COV504, and OV56), Health Science Research Resources Bank (KURAMOCHI and OVSAHO), Sigma (COV664), DSMZ (EFO-21), Riken (JHOS-2) and Korean Cell Line Bank (SNU-119).
Pleural fluid containing CA-125 at 183,600 U/ml was purchased from ADVY Chemical (Mumbai, India). CellTiter-Glo® reagent was from Promega (Mannheim, Germany). 293Fectin, cell culture medium, 6-carboxy-fluorescein succinimidyl ester (CFSE), and pcDNA 3.1 vector were obtained from Thermo Fisher Scientific (Darmstadt, Germany). For surface plasmon resonance (SPR) measurements, CM5 Sensor Chips, Amine Coupling Kit, Human Antibody Capture Kit, and 10x HBS-N-buffer from GE Healthcare were used (Dornstadt, Germany). PBS 10x, Tween 20, and BSA were from Roche (Sigma-Aldrich distributor; Taufkirchen, Germany). RG7787 has been previously described26 (link) and was produced at Roche from inclusion bodies by refolding37 (link).
If not otherwise indicated, all experiments were repeated at least twice.
Pleural fluid containing CA-125 at 183,600 U/ml was purchased from ADVY Chemical (Mumbai, India). CellTiter-Glo® reagent was from Promega (Mannheim, Germany). 293Fectin, cell culture medium, 6-carboxy-fluorescein succinimidyl ester (CFSE), and pcDNA 3.1 vector were obtained from Thermo Fisher Scientific (Darmstadt, Germany). For surface plasmon resonance (SPR) measurements, CM5 Sensor Chips, Amine Coupling Kit, Human Antibody Capture Kit, and 10x HBS-N-buffer from GE Healthcare were used (Dornstadt, Germany). PBS 10x, Tween 20, and BSA were from Roche (Sigma-Aldrich distributor; Taufkirchen, Germany). RG7787 has been previously described26 (link) and was produced at Roche from inclusion bodies by refolding37 (link).
If not otherwise indicated, all experiments were repeated at least twice.
9
Comprehensive Protein Sample Preparation
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Acetonitrile, deoxycholic acid (DC), NaCl and ponceau S were from Sigma (Sigma-Aldrich, Steinheim, Germany), and bromophenol blue, Coomassie Bue Brilliant R-250 and FA from Fluka (Fluka Chemie, Buchs, Switzerland). Tween-20 was bought from Roche Diagnostics (Mannheim, Germany). DTE, glycerol and urea were purchased from MP Biomedicals (Illkirch, France), ammonium bicarbonate and iodoacetamide from ICN Biomedicals (Aurora, Ohio, United States), 0.9% NaCl from Baxter (Volketswil, Switzerland), 10x PBS (1x PBS eq. to 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4 and 1.8 mM KH2PO4) from Laboratorium Dr. G. Bichsel (Interlaken, Switzerland), EDTA from Merck (MSD Merck Sharp & Dohme, Luzern, Switzerland), Tris-HCl from BIO-RAD (Hercules, CA, United States), ethanol from Thommen-Furler AG (Rüti bei Büren, Switzerland), Top Block from Lubio Science (Luzern, Switzerland), benchMark Protein Ladder (prestained or not) from Invitrogen (Carlsbad, CA, United States), FITC mouse anti-Human CD47 antibody from BD Pharmingen (BD Biosciences, Franklin Lakes, NJ, United States), and Sequencing Grade Modified Trypsin (Trypsin) from Promega AG (Dübendorf, Switzerland). Deionized water (18.2 MΩ⋅cm) was prepared using a Purelab option Q-15 (Elga LabWater).
10
Quantifying Osteogenic Differentiation via ALP
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The cells were fixed on days 3, 7, 14, 21, and 28 after incubation with an osteogenic differentiation medium, and ALP staining was performed using the following methods. After fixation with 4% paraformaldehyde PBS (Fujifilm Wako Pure Chemical, Osaka, Japan) for 10 min, the cells were incubated with PBS containing 0.05% Tween-20 (Roche Diagnostics) (washing buffer). After removing the washing buffer, the cells were incubated with ALP staining solution (Fujifilm Wako Pure Chemical Industries, Ltd.) at room temperature under light-resistant conditions for 10 min.
For the quantitative testing of ALP, the cells were harvested on days 3, 7, 14, 21, and 28 using the pNPP Phosphatase Assay Kit (AnaSpec, Fremont, CA, USA) after incubation with an osteogenic differentiation induction medium. The harvested cells were homogenized using a Sonic Vibra Cell (Sonic & Materials, Newtown, CT, USA), and the supernatant was collected and used as the sample. The sample was mixed with 50 μL of the pNPP substrate solution in a 96-well plate (CORNING), and the absorbance was determined at a wavelength of 405 nm using a microplate reader MultiskanTM FC (Thermo Fisher Scientific).
For the quantitative testing of ALP, the cells were harvested on days 3, 7, 14, 21, and 28 using the pNPP Phosphatase Assay Kit (AnaSpec, Fremont, CA, USA) after incubation with an osteogenic differentiation induction medium. The harvested cells were homogenized using a Sonic Vibra Cell (Sonic & Materials, Newtown, CT, USA), and the supernatant was collected and used as the sample. The sample was mixed with 50 μL of the pNPP substrate solution in a 96-well plate (CORNING), and the absorbance was determined at a wavelength of 405 nm using a microplate reader MultiskanTM FC (Thermo Fisher Scientific).
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