Porcine pepsin (P7012; 2971 IU/mg), porcine pancreatin (P7545; 6.79 IU/mg), bovine bile extract (B8631; 3.1 mmol/g), and the enzyme inhibitors pepstatin A (P5318) and pefabloc (76,307) were all obtained from Sigma-Aldrich (St. Quentin Fallavier, France). Bovine bile salts concentration and enzyme activities were determined as described in the Electronic Supplementary Information of [39 (link)]. The fluorescent dyes used for Confocal laser scanning microscopy (CLSM) analysis were high-content screening (HCS) LipidTOX™ Green Neutral Lipid Stain (H34475) obtained from Thermo Fisher Scientific (Illkirch, France), Rd-DOPE® Liss Rhod PE 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (810150C), and Fast Green Food Green 3 (FCF) (Sigma F7258, Sigma-Aldrich, St Louis, MO, USA). The standards used for size exclusion chromatography were purchased from Phenomenex (Waters Inc., Milford, MA, USA) (No. ALO-3042 for bovine thyroglobulin, IgA, IgG, ovalbumin and myoglobin) and from Sigma-Aldrich (cytochrome C (C2506) and human angiotensin II (A9525)). All other chemicals were of standard analytical grade.
Lipidtox green neutral lipid stain
Manufactured by Thermo Fisher Scientific
Sourced in United States
LipidTOX™ Green Neutral Lipid Stain is a fluorescent dye that specifically binds to neutral lipids in cells. It can be used to detect and quantify intracellular lipid droplets.
Automatically generated - may contain errors
Lab products found in correlation
Porcine pancreatin, by Merck Group (1 mentions)
Bovine bile extract, by Merck Group (1 mentions)
Pefabloc, by Merck Group (1 mentions)
Pepstatin a, by Merck Group (1 mentions)
Porcine pepsin, by Merck Group (1 mentions)
Anti dsred, by Takara Bio (1 mentions)
Ab181595, by Abcam (1 mentions)
Ab150075, by Abcam (1 mentions)
Ab18586, by Abcam (1 mentions)
Vectamount mounting medium, by Vector Laboratories (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Tcs sp8, by Leica (1 mentions)
Lamp1 h4a3, by Developmental Studies Hybridoma Bank (1 mentions)
Inverted microscope, by Leica (1 mentions)
Tcs spe, by Leica (1 mentions)
Powershot digital camera, by Canon (1 mentions)
Prolong diamond antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
Chloroform, by Avantor (1 mentions)
11 protocols using lipidtox green neutral lipid stain
1
Porcine Pepsin and Pancreatin Characterization
2
Visualizing Brown Adipose Tissue
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Mice were transcardially perfused with PBS. BAT was fixed overnight at 4 °C with 4% paraformaldehyde and was cut 50 μM sections with a vibratome. The sections were incubated with 5% bovine serum albumin (BSA) at room temperature and then with anti-UCP1 (1:1000, Santa Cruz biotechnology, Inc., sc-6529) and anti-DsRed (1:1000, Clontech laboratories, Inc., 632496) antibodies in 0.2% Triton X-100 overnight at 4 °C. Following incubation with primary antibodies, the sections were washed 3 times in PBS and incubated with Alexa 488 anti-goat IgG (1:500, A11055) and Alexa 568 anti-rabbit IgG (1:500, Thermo Fisher Scientific, Inc., A10042) for 2 hr at room temperature. For lipid staining, tissues were incubated with LipidTox Green neutral lipid stain (1:200, Thermo Fisher Scientific, Inc., H34475) for 30 min at room temperature. Tissues were then washed, dried, and mounted with VECTASHIELD mounting media. Images were acquired using a scanning confocal microscope.
3
Immunostaining of Human Eyelid Sections
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Human eyelid sections and HMGECs were fixed with cold methanol for 15 minutes at −20°C. Following three phosphate‐buffered saline (PBS) rinses for 5 minutes each, samples were blocked with 2% bovine serum albumin (BSA, Sigma‐Aldrich Corp., St. Louis, MO) in PBS for 60 minutes, and then incubated overnight at 4°C in a moist chamber with antibodies specific for Lrig1 (ab214102,1:100), cytokeratin 14 (ab181595, 1:500), cytokeratin 6 (ab18586, 1:500), DNase2 (ab8119, 1:100; Abcam, Cambridge, MA), or lysosomal‐associated membrane protein 1 (LAMP‐1;H4A3, 1:15; Developmental Studies Hybridoma Bank, Iowa City, IA), or the BSA diluent. After three additional PBS rinses, donkey anti‐rabbit (ab150075, 1:500, Abcam) or donkey anti‐mouse (2492098, 1:500, EMD Millipore, Temecula, CA) secondary antibodies were applied for 1 hour at room temperature. For neutral lipid staining, some eyelid sections were fixed in 4% paraformaldehyde for 15 minutes. Following additional washes, samples were exposed to LipidTOX Green neutral lipid stain (1:500, Thermo Fisher Scientific) in a humid chamber for 30 minutes. Nuclei were counterstained with 4′,6‐diamidino‐2‐phenylindole (DAPI, 1 μg/mL, Sigma‐Aldrich) and samples were covered with VectaMount mounting medium (Vector Laboratories, Burlingame, CA), and observed with a confocal microscope (Leica TCS SP8, Leica Microsystems, Wetzlar, Germany).
4
Multiparameter Flow Cytometry Assay
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Isolated cells were suspended in DPBS-/- containing 0.02% bovine serum albumin and 1% Fc-blocking IgG (v/v) and incubated with 0.1% LIVE/DEAD Fixable Stain for 20 min on ice. Excess stain was removed, and cells incubated with fluorescently labeled antibodies for 30 min on ice. Cells were washed and then fixed using 1% paraformaldehyde. In select experiments, LipidTOX Green Neutral Lipid Stain (ThermoFisher) was added to cells at 1× and incubated for 30 min. CountBright Absolute Counting Beads were used for leukocyte enumeration. Staining was evaluated using LSRFortessa cell analyzer and analyzed using FlowJo software.
5
Visualizing Cellular Lipid Accumulation
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To visualize the accumulation of lipid droplets within the cells after PA treatment, they were stained with Oil Red O dye. Prior to staining, cells were washed with phosphate buffer saline (PBS) and fixed with 4% paraformaldehyde (PFA). An additional wash was performed to remove the fixation solution, and cells were treated with 60% isopropanol for 5 min. After the removal of alcohol and washing, cells were incubated with Oil Red O solution for 15 min at room temperature (RT). Specimens were observed under an inverted microscope (Leica), and pictures were acquired using a Canon PowerShot digital camera. Accumulation of dye was quantified using spectrophotometrical measurement. Briefly, the accumulated dye was washed out from the cells using 100% isopropanol and subjected to 96-well plates for the measurement of absorbance at 490 nm/570 nm wavelengths using a plate reader (Epoch, Biotek, Bad Friedrichshall, Germany).
Alternatively, neutral lipid accumulation was observed in cells using Lipid Tox™ Green Neutral Lipid Stain (ThermoFisher). Fixed samples were incubated with the dye solution (1:200) for 30 min at RT. After washing, the dye, the specimens were mounted on glass slides using ProLong™ Diamond Antifade Mountant with DAPI (ThermoFisher) and observed in a confocal microscope (Leica TCS SPE).
Alternatively, neutral lipid accumulation was observed in cells using Lipid Tox™ Green Neutral Lipid Stain (ThermoFisher). Fixed samples were incubated with the dye solution (1:200) for 30 min at RT. After washing, the dye, the specimens were mounted on glass slides using ProLong™ Diamond Antifade Mountant with DAPI (ThermoFisher) and observed in a confocal microscope (Leica TCS SPE).
6
Lipid Staining in Cells and Tissues
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For lipid staining in cell culture, cells grown on glass chamber slides (Nalgene Nunc International, Naperville, IL, USA) were washed with PBS and fixed in 4% paraformaldehyde for 30 min. Slides were incubated in LipidTOX Green neutral lipid stain (Invitrogen, Grand Island, NY, USA) in a moist chamber for 30 min at room temperature. Slides were then mounted in VectaShield mounting medium containing DAPI (Vector Laboratories), and observed under a fluorescence microscope. Quantification of lipid staining was performed using imageJ software (NIH, Bethesda, MD, USA). For lipid staining in mouse tissue, cryosections (10-μm thick) were air-dried, rinsed in 60% isopropanol, and stained with 0.3% Oil Red O (Fisher Biotec, Wembley, WA, Australia) for 15 min at room temperature. The sections were rinsed again and mounted in VectaShield mounting medium for light microscopy.
7
Quantitative Analysis of Cellular Lipids
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All solvents and chemicals were obtained from Merck (Dorset, U.K.) unless otherwise stated. All cell culture medium was obtained from Gibco (U.K.) unless otherwise stated. 4–12% Bis-Tris gels, first-strand buffer (Invitrogen), SuperScript II Reverse Transcriptase, TRIzol, LipidTOX green neutral lipid stain, TGF beta-1 enzyme-linked immunosorbent assay (ELISA) Kit and Click-iT (5-ethynyl-2′-deoxyuridine) EdU Alexa Fluor 488 HCS Assay were obtained from Invitrogen. Tris-base, acetate, chloroform, EDTA, glycerol, KCl, NaOH, Tris–HCl and Tween-20 were obtained from VWR (West Sussex, U.K.). SensiMix SYBR Hi-ROX was obtained from Bioline (London, U.K.).
8
Antibody-Based Protein Analysis for AKT Signaling
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Antibodies against AKT and phospho-AKT (Ser473) were from Cell Signaling (Danvers, MA). Anti-GAPDH and horseradish peroxidase–linked secondary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Glucose, Glucose oxidase, collagenase, and RPMI medium were from Sigma-Aldrich (St. Louis, MO). Free FA (FFA) and TG assay kits ware purchased from Wako Diagnostics (Richmond, VA). The LipidTOX green neutral lipid stain, NuPAGE gels, SuperScript III reverse transcriptase, and oligo(dT)12–18 primer were from Invitrogen (Carlsbad, CA). The ultrasensitive mouse insulin ELISA kit was from ALPCO (Salem, NH). The anti-insulin and anti-glucagon antibodies were from Abcam (Cambridge, MA). HF diet (60 kcal% from fat; catalog number D12492) was from Research Diets (New Brunswick, NJ). The control diet (CD) (17 kcal% from fat, catalog number 7912) was from Harlan Laboratories (Madison, WI).
9
Lipid Staining for Adipogenic Differentiation
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To avoid no-specific signal, LipidTOX™ Green neutral lipid stain (Invitrogen, Molecular Probes, Grand Island, NY, USA) was used to assess the adipogenic differentiation on INTEGRA® [40 (link)]. Cells were fixed in 4% paraformaldehyde for 10 min at room temperature. After initial washing with PBS and a final wash with H2Od, cells were stained with 250 L H2Od, cell LipidTOX™ Green neutral lipid stain solution for 20 min at room temperature. Stained samples were mounted and nuclei were counterstained with Vectashield with diamidino-2-phenylindole (DAPI) (Vector Laboratories Inc., Burlingame, CA, USA). Images were acquired using fluorescence microscopy (Eclipse-TE2000-S, Nikon, Düsseldorf, Germany) using the F-ViewII FireWire™ camera (Olympus Soft Imaging System, Münster, Germany).
10
Adipogenic Differentiation Quantification
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Oil red O (Solarbio, G1260) or neutral lipid droplet staining was performed to confirm adipogenic differentiation. Samples were fixed in 4% paraformaldehyde for 30 min. For Oil red O staining, samples were treated with a solution of Oil red O and water (2:3) for an additional 30 min before rinsing twice with water. Images were captured under brightfield using a Zeiss microscope. To assess the size of adipogenic cells, the incorporated Oil red O dye was extracted in a culture dish with 1 mL isopropanol by shaking for 5 min at 25°C. One hundred microliters of the medium were collected and the absorbance was read using a multi-scan spectrum at 492 nm. LipidTOX Green neutral lipid stain (Molecular probes, H34475, 1:200) was added to the samples for 45 min to stain neutral lipid droplets and images were captured using a Zeiss LSM 800 laser confocal microscope. Quantitative analysis of the lipid droplets was performed using ImageJ, which was used to measure the diameters of the lipid droplets in each image. The proportion of lipid droplets in each diameter was then estimated.
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