Cortical granule (CG) lectin was stained as described previously.22 , 23 Oocytes were fixed with 4% paraformaldehyde (Wako Pure Chemical Industries, Ltd, Osaka, Japan) in Dulbecco's phosphate‐buffered saline (PBS) for 1 h at room temperature, followed by incubation in blocking solution, which comprised PBS containing 0.2% polyvinyl alcohol (PVA; Sigma‐Aldrich), 0.3% bovine serum albumin (BSA) (Sigma‐Aldrich), and 100 mM glycine (Kanto Chemical, Tokyo, Japan) for 5 min. Subsequently, oocytes were washed with PBS/PVA containing 0.1% Triton X‐100 (Wako Pure Chemical Industries, Ltd) and incubated with Alexa Fluor 488‐conjugated lectin PNA (1:200; Molecular Probes, OR) for 30 min at room temperature. After washing in PBS/PVA containing 0.01% Triton X‐100 and 0.3% BSA, oocytes were mounted onto glass slides using VECTASHIELD Mounting Medium (Funakoshi, Tokyo, Japan). Images were obtained using a TCS SP5 confocal laser microscope (Leica, Tokyo, Japan).
Triton x 100
Manufactured by Fujifilm
Sourced in Japan, United States
Triton X-100 is a non-ionic surfactant commonly used in laboratory applications. It is a clear, colorless liquid with a viscous consistency. Triton X-100 is designed to solubilize membrane proteins and lipids, making it a useful tool for various biochemical and analytical procedures.
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Lab products found in correlation
Bovine serum albumin (bsa), by Merck Group (15 mentions)
Paraformaldehyde (pfa), by Fujifilm (10 mentions)
Hoechst 33342, by Thermo Fisher Scientific (9 mentions)
Bovine serum albumin (bsa), by Fujifilm (6 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Lsm 880, by Zeiss (6 mentions)
Propidium iodide, by Fujifilm (4 mentions)
Tween 20, by Fujifilm (4 mentions)
Propidium iodide, by Thermo Fisher Scientific (4 mentions)
Rnase a, by Thermo Fisher Scientific (4 mentions)
Lsm 700, by Zeiss (4 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (4 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (4 mentions)
Triglyceride e test, by Fujifilm (3 mentions)
Bio plex assay system, by Bio-Rad (3 mentions)
Multi beads shocker, by Yasui Kikai (3 mentions)
Hoechst 33342, by Dojindo Laboratories (3 mentions)
Fluorescence mounting medium, by Agilent Technologies (3 mentions)
Sodium chloride (nacl), by Fujifilm (3 mentions)
Bz x700, by Keyence (3 mentions)
129 protocols using triton x 100
1
Cortical Granule Lectin Staining in Oocytes
2
Antiparasitic Efficacy Screening Assay
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Host 3T3 cells were seeded onto 96-well black, clear bottom microplate (Corning Inc., USA) at 5 × 103 cells/well in 100 μL of DMEM supplemented with 10% FBS for 4 h to allow cell attachment. Tissue-derived trypomastigotes were added at multiplicity of infection (MOI) of 20, and incubated at 37°C for 24 h. Uninfected trypomastigotes were removed by washing with PBS, followed by addition of 100 μL of DMEM (10% FBS) containing benznidazole or nifurtimox dissolved in DMSO. The final concentration of DMSO was 0.5%. After 48 h incubation at 37°C, the media was removed from wells and cells were fixed by addition of 100 μL of 4% formaldehyde for 15 min at room temperature. After fixation, the nuclei were stained by adding 100 μL of 1.0 μg/mL Hoechst 33342 (Thermo Fisher Scientific) and 0.05% Triton X-100 (Wako Pure Chemical Industries, Ltd., Japan) for 15 min, followed by washing wells with PBS 4 times. The plates were imaged by a fluorescence microscopy. Host cells containing more than three amastigotes were considered as infected. EC50 was calculated by fitting the dose response curves with non-linear regression analysis, using “(inhibitor) vs. normalized response" model of GraphPad Prism7 software.
3
Immunohistochemical and Imaging Analysis of Lung Tissue
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Immunohistochemical staining was performed as previously described with minor modification on the paraffin-embedded lung tissues.7,45 (link) PARK2 immunostaining were assessed by counting total and positively staining cells in small airways at a magnification of ×400 and percentage of positive staining was scored in a semi-quantitative fashion; 0 (less than 10%), 1 (11 to 49%), and 2 (more than 50%). Immunofluorescence staining was also performed as previously described.7,45 (link) BEAS-2B cells expressing EGFP-LC3B grown on 8-well culture slides were fixed with 4% paraformaldehyde for 15 min followed by permeabilization with 0.03% Triton X-100 (Wako, 160-24751) for 60 min. After blocking with 0.1% BSA (Sigma-Aldrich, A2153) for 60 min, the primary and secondary antibodies were applied according to the manufacturer's instructions. Confocal laser scanning microscopy analysis of mitochondria was performed by TOMM20 staining, assessed with a confocal microscope (Carl Zeiss LSM510, Tokyo, Japan). Fluorescence microscopy analysis of phospho-histone H2AFX, and TOMM20 staining were performed in HBEC (Olympus, Tokyo, Japan). MitoTracker Red CMXRos (MTR; Molecular Probes-Life Technologies, M-7512) staining (200 nM, 30 min at 37°C) was also performed to evaluate the integrity of the mitochondrial membrane potential.
4
Cellular DNA Content Quantification
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Quantification of cellular DNA content at 72 h after transfection with control miRNA or #12 (5 nM) was determined by using a cytometer. Briefly, the cells were harvested and fixed with 70% cold ethanol at −20°C overnight. The fixed cells were then washed twice with PBS and resuspended in 100 μL PBS-based propidium iodide solution containing 0.1% Triton X-100 (Wako, Osaka, Japan), 0.2 mg/mL RNase A (Life Technologies), and 20 μg/mL propidium iodide (Life Technologies). Thereafter, they were incubated for 30 min at room temperature protected from the light. The DNA content in the cells was analyzed with a cytometer (The Tail Image-Based Cytometer, Life Technologies).
5
Decellularization of Porcine Liver
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Decellularization was performed as described previously [14] (link). Briefly, a healthy porcine liver was harvested from adult pig (20–25 kg) (Fukuokashokunikuhanbai Co., Ltd., Fukuoka, Japan) and depleted of blood with calcium and magnesium-free phosphate-buffered saline (CMF-PBS). The liver was cut into 1 cm × 1 cm × 2 mm pieces using a mandoline-style slicer. The sliced tissue was soaked in a solution containing 1% Triton X-100 (Wako Pure Chemical Industries, Ltd., Osaka, Japan) in CMF-PBS at 4 °C for 3 days to remove the cellular components. Fresh solution was added each day, for 3 days, under constant stirring to maintain the efficiency of decellularization. The resulting decellularized liver was immersed in CMF-PBS at 4 °C for 4 days, and then dialyzed using the Spectra/Por 6 dialysis membrane (MWCO: 1000 kD, Spectrum Laboratories, Inc., Milpitas, CA, USA) at 4 °C for 2 days.
6
Quantification of HSP70 Expression in Breast Cancer Cells
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MDA-MB-231 and MDA-MB-453 cells were treated with EPI (10 μM) for 30, 60, and 180 min and fixed with 4% paraformaldehyde phosphate-buffered solution (Fujifilm Wako), followed by permeabilization with 0.1% Triton X-100 (Fujifilm Wako) in PBS. After blocking with 10% FBS in PBS, the cells were incubated overnight with HSP70 primary antibody (Table S1 ) at 4 °C. The cells were incubated with Alexa Fluor 594-AffiniPure anti-mouse IgG (1:100, cat. no. 115-585-003, Jackson Immuno Research, West Grove, PA, USA) and 0.5 μg/mL DAPI (Fujifilm Wako) for the counterstaining of nuclei. The fluorescence was visualized using an Olympus FSX100 microscope (Olympus, Tokyo, Japan). The fluorescence intensity of HSP70 was analyzed using Image J 1.52a software.
7
Immunocytochemistry and Immunohistochemistry of Muscle Tissue
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For immunocytochemistry, satellite cells associated with myofibers were fixed in 2% paraformaldehyde for 10 min at room temperature. Samples were then incubated with primary antibodies at 4 °C overnight following blocking/permeabilization with phosphate-buffered saline containing 0.3% Triton X-100 (Wako Pure Chemical Industries Ltd., Osaka, Japan) and 5% goat serum for 20 min at room temperature. For immunohistochemistry, muscle tissues were isolated from the tibialis anterior (TA) muscle, immediately frozen in 2-methylbutane cooled in liquid nitrogen, and stored at −80 °C. Frozen cross-sections of the TA muscle were fixed in 4% paraformaldehyde and blocked with a DAKO blocking reagent (DAKO Japan, Kyoto, Japan), and incubated with primary antibodies at 4 °C overnight. Immunostained samples were visualized using appropriate species-specific Alexa Fluor 488 or 568 fluorescence-conjugated secondary antibodies (Thermo Fisher Scientific, Tokyo, Japan). Samples were viewed on an all-in-one fluorescence microscope (Keyence, Osaka, Japan) or an Olympus IX83 microscope (Olympus, Tokyo, Japan). Images were optimized globally and assembled into figures using Adobe Photoshop CS5.1 (Adobe Systems Inc., San Jose, CA, USA).
8
Cytotoxicity of Caffeic Acid, CAPE, and EGCG on KN-3 cells
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KN-3 cells seeded in 24-well plates were treated with caffeic acid, CAPE, or EGCG in the concentration of 0.1 to 10 μg/ml for 24 hours. Cytotoxic effect of caffeic acid, CAPE, and EGCG on the viability of KN-3 cells was assessed by observing the cell morphology under the microscope and quantified the amount of LDH released into the culture supernatant using LDH Cytotoxicity Assay Kit (Cayman Chemical, Ann arbor, MI, USA). Treatment with 0.1% Triton X-100 (Wako) for 10 minutes was used as a positive control.
9
Mouse Serum and Liver Lipid Analysis
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Total, low-density lipoprotein (LDL), and high-density lipoprotein (HDL)-cholesterol, triglycerides and free fatty acids were measured in mouse serum using Hitachi 7180 auto-analyzers at the Nagahama Research Institute (Oriental Yeast Co., Ltd., Shiga, Japan). Enzyme-linked immunosorbent assay (ELISA) was used to measure serum insulin (mouse insulin ELISA kit, Shibayagi Co., Ltd., Gunma, Japan) and sIgA concentrations (ELISA Kit for Secretory Immunoglobulin A, Cloud-Clone Corp., Texas, USA). A 2:1 (v/v) chloroform-methanol solution was used to extract lipids from the liver [21 (link)], which were then dissolved in isopropanol containing 10% polyoxyethylene octylphenyl ether (Triton X-100, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). Cholesterol and triglyceride concentration in the lipid extracts were measured enzymatically using the Cholesterol E-test and Triglyceride E-test, respectively (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan).
10
Metabolic Biomarkers in Mice
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Total, LDL, and HDL cholesterol, triglycerides and non-esterified fatty acids (NEFA) were measured in mouse sera using Hitachi 71,870 auto-analyzers at the Nagahama Research Institute (Oriental Yeast Co., Ltd., Shiga, Japan). Serum leptin and insulin concentrations were measured using enzyme-linked immunosorbent assay (ELISA) kits (Mouse Leptin Immunoassay Kit, R&D Systems, Inc., MN, USA; Mouse Insulin ELISA Kit (TMB), Shibayagi Co., Ltd., Gunma, Japan). A 2:1 (v/v) chloroform–methanol solution was used to extract lipids from the liver [26 ], which were then dissolved in isopropanol containing 10% Triton X-100 (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). Hepatic cholesterol and triglyceride levels were measured enzymatically with commercial kits (Cholesterol E-test and Triglyceride E-test; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan).
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