The chip device was taken out of the incubator for fixation, and the lid was carefully removed in a sterile biosafety cabinet. The kidney organoids adherent on the surface of the ECM were rinsed with PBS, carefully overlaid with 4% PFA, and fixed for 45 min at RT. After fixation, the samples were washed three times with 0.1% Triton X-100 (Sigma-Aldrich) for 30 min. Organoids were stained and cleared with ethyl cinnamate (Sigma-Aldrich), as described before (78 (link)). The following antibodies were used in these studies: anti-CDH1 (Abcam, ab11512), anti-CFTR (Sigma-Aldrich, HPA021939), anti-LTL (Vector Laboratories, B-1325), anti-PODXL (R&D Systems, AF1658), anti-KI67 (Dako, m7248), anti-pS6RP (Cell Signaling Technology, 2211S), anti-RAC1 (Developmental Studies Hybridoma Bank, CPTC-RAC1-2), and anti-FOS (Abcam, ab190289). Samples were examined with a Zeiss TIRF/LSM 710 confocal microscope or a Leica STELLARIS 8 confocal microscope and by using Imaris 3D software (Bitplane).
Ethyl cinnamate
Manufactured by Merck Group
Sourced in United States
Ethyl cinnamate is a colorless, oily liquid chemical compound used in various laboratory applications. It has a pleasant, sweet, and floral aroma. Ethyl cinnamate serves as a chemical reagent and flavoring agent in research and industrial settings.
Automatically generated - may contain errors
Lab products found in correlation
Eugenol, by Merck Group (3 mentions)
Benzaldehyde, by Merck Group (2 mentions)
Ultramicroscope 2, by Miltenyi Biotec (2 mentions)
Eosin y, by Leica (2 mentions)
Ethyl acetate, by Merck Group (2 mentions)
Acetic acid, by Merck Group (2 mentions)
Triton x 100, by Merck Group (1 mentions)
Anti p s6rp, by Cell Signaling Technology (1 mentions)
Anti ki67, by Agilent Technologies (1 mentions)
Ab190289, by Abcam (1 mentions)
B 1325, by R&D Systems (1 mentions)
Benzyl alcohol, by Merck Group (1 mentions)
Propylene glycol, by Thermo Fisher Scientific (1 mentions)
Vanillin, by Merck Group (1 mentions)
Triacetin, by Merck Group (1 mentions)
P anisaldehyde, by Merck Group (1 mentions)
L menthol, by Merck Group (1 mentions)
Maltol, by Merck Group (1 mentions)
Ethyl maltol, by Merck Group (1 mentions)
Sp8 2 photon microscope, by Leica (1 mentions)
25 protocols using ethyl cinnamate
1
Kidney Organoid Fixation and Imaging
2
Evaluation of E-Cigarette Refill Fluids
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Thirty-nine commercial EC refill fluids and duplicate bottles of six of these products (45 total products) were purchased from online vendors including Freedom Smoke USA (Tucson, AZ), Global Smoke (Los Angeles, CA), Johnson Creek (Johnson Creek, WI), Red Oak (a subsidiary of Johnson Creek), Tasty Puff (Albuquerque, NM), e-cigexpress (Orlando, FL), Vaporbomb.com (Barberton, OH), Vapormaxx (Richmond, VA), and DIY Flavor Shack (Las Vegas, NV). Inventory number, flavor name, and company are given in Supplemental Table 1 and details of each product have been previously published4 (link)–6 . Commercial fluids were stored in 4 °C and cytotoxicity did not change with storage6 .
Authentic standards of chemicals: (1) trans-cinnamaldehyde from TCI (Tokyo, Japan), (2) L-menthone and hydroxyacetone from Alfa Aesar (Ward Hill, MA, USA), (3) ethyl maltol, L-menthol, maltol, benzyl alcohol, ethyl cinnamate, eugenol, p-anisaldehyde, triacetin, vanillin, and benzaldehyde from Sigma-Aldrich (St. Louis, MO, USA), and (4) propylene glycol from Acros Organics (New Jersey, USA). triacetin is a flavor chemical as well as a solvent. Each authentic standard was dissolved in propylene glycol (80%) and distilled water (<20%) to simulate hand-made refill fluids. A propylene glycol control blank was prepared with 80% propylene glycol and 20% distilled water.
Authentic standards of chemicals: (1) trans-cinnamaldehyde from TCI (Tokyo, Japan), (2) L-menthone and hydroxyacetone from Alfa Aesar (Ward Hill, MA, USA), (3) ethyl maltol, L-menthol, maltol, benzyl alcohol, ethyl cinnamate, eugenol, p-anisaldehyde, triacetin, vanillin, and benzaldehyde from Sigma-Aldrich (St. Louis, MO, USA), and (4) propylene glycol from Acros Organics (New Jersey, USA). triacetin is a flavor chemical as well as a solvent. Each authentic standard was dissolved in propylene glycol (80%) and distilled water (<20%) to simulate hand-made refill fluids. A propylene glycol control blank was prepared with 80% propylene glycol and 20% distilled water.
3
Imaging Neonatal Mouse Lungs
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Lungs from P7 neonatal mice treated with PBS or BMP9 were perfused with heparinized PBS before harvest and inflation via the trachea with 0.75% Phytagel (Sigma Aldrich). Lungs were then fixed overnight at 4 °C in 4% PFA and dehydrated by sequential overnight incubations in 50% ethanol at pH 9, 70% ethanol at pH 9, and 2 nights at 100% ethanol, respectively. Following dehydration, tissues were incubated overnight in ethyl cinnamate (Sigma Aldrich) for clarification before imaging on a Leica SP8 2-photon microscope.
4
Intravital Imaging of Lung Vasculature
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Naïve or 4 days following PR8 infection (I.N. or I.T.) mice were intravitally stained for VCAM-1+ and ICAM-1+ lung vessels by I.V. injection of 5 μg of Alexa-647 anti-VCAM-1 (CD106) mAb (BD Bioscience, Cat. 561612, clone 429 (MVCAM.A)) or Alexa-568 conjugated anti-ICAM-1(CD54) mAb (Biolegend, Cat. 116101, clone YN1/1.7.4) 5 minutes prior to euthanasia. Mice were transcardially perfused with PBS and the lungs were inflated via the trachea with low-gelling agarose (Sigma Aldrich, Cat. A9045), fixed with paraformaldehyde (4% in PBS) for 2 hours, dehydrated, and cleared using ethyl cinnamate (Sigma Aldrich, Cat. 112372) as described (18 (link)). Cleared lung lobes were imaged in an Ultramicroscope II (LaVision BioTec) operated by the ImspectorPro software (LaVision BioTec, Bielefeld, Germany) as described (18 (link)). For the visualization of PR8 mice were I.T. infected with 3x103 PFU of PR8-mCherry and lungs were imaged 4 days later. The three-dimensional rendering of LSM was performed via Imaris software (Oxford Instruments, Abingdon, UK).
5
Profiling Ginger Phytochemicals from Diverse Sources
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Twenty ginger samples from different origins were purchased from different vendors through Amazon.com, and two ginger samples were collected from Sagamu and Zaria districts of Nigeria (details provided in Supplementary Table S1 ). All samples were in the form of dry powder. Six-gingerol (6G, ≥ 98% purity), 8-gingerol (8G, ≥ 95% purity), 10-gingerol (10G, ≥ 95% purity), ethyl cinnamate, cinnamyl acetate, 8-shogaol, and MTT were purchased from Sigma Aldrich (St. Louis, MO, USA). Six-paradol was purchased from Adooq (Irvine, CA, USA), while pinolenic acid was sourced from Santa Cruz (Dallas, TX, USA). Deionized water was prepared using a Milli-Q purification system (Millipore, Billerica, MA, USA). LC–MS grade acetonitrile, methanol, and formic acid were purchased from Sigma Aldrich (St. Louis, MO, USA). Ethanol was purchased from Decon Laboratories, Inc. (King of Prussia, PA, USA). Dimethyl sulfoxide (DMSO) was purchased from Fisher Scientific (Pittsburgh, PA, USA).
6
Lung-Accumulated Human Blood ILC2 Visualization
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For light-sheet microscopic visualization of lung-accumulated human blood ILC2s, the lung tissue was prepared as described elsewhere (50 (link)). In short, lung lobes were fixed in 4% PFA for 2 h at 4°C under constant rotation. Dehydration was performed in an ascending ethanol series of 50, 70, and 100% (2x) for at least 4 h each. Dehydrated samples were then cleared with ethyl cinnamate (Sigma) at room temperature and imaged with the UltraMicroscope II (LaVision, BioTec). 3D reconstruction and quantification of accumulated human blood ILC2s within the pulmonary tissue were performed with the Imaris Image Analysis software 9.0.2 (Bitplane) as described in detail earlier (49 (link)). Accumulated labeled cells were counted in 2 to 3 lung cubes with defined volume per lung with a mean cell count of 156 labeled ILC2s per lung cube.
7
Fluorescent Nuclear Staining and Optical Tissue Clearing
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Tissue samples were stained with a fluorescent nuclear stain, TO-PRO-3 (Cat: T3605, Thermo-Fisher) at a 1:2000 dilution and eosin (Cat: 3801615, Leica Biosystems) at a 1:2000 dilution for 4 hours at room temperature with light shaking. Samples were optically cleared with ethyl-cinnamate (Cat: 112372, Sigma-Aldrich). Stained and cleared samples were imaged on a custom OTLS system [14 (link)]. A 660-nm laser was used to excite the nuclear dye, TO-PRO3, and a 561-nm laser was used to excite eosin. Each channel was imaged separately, in succession, with a 16-bit sCMOS camera. For more information on OTLS imaging see our previous publications [12 (link)–14 (link)].
8
Clearing and Imaging of PFA-Fixed HH25 Embryos
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PFA-fixed HH25 embryos were cleared using an adapted Ethyl-Cinnamate protocol (Klingberg et al., 2017 (link)). Briefly, tissues were dehydrated in ethanol successive baths finally cleared in Ethyl Cinnamate (Sigma, 112372). Cleared samples were imaged using the UltraMicroscope SPIM (LaVision Biotech). 3D-images were built using ImarisTM software. Volumetric analysis was performed using ImarisTM “Surface” module adjusted on CFSE fluorescence.
9
Phenylpropanoids Profiling for Pest Control
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Eleven phenylpropanoids, including α-asarone, eugenol, isoeugenol, methyl eugenol, methyl isoeugenol, coumarin, coumarin 6, coniferyl aldehyde, diniconazole, ethyl cinnamate, and rosmarinic acid, based on their toxicity to other pest species as shown in Figure 3 were purchased from Sigma Aldrich, London, UK.
10
Optical Clearing of Bone Samples
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