Cells were washed in phosphate-buffered saline (PBS) and then fixed in ice-cold methanol at −20°C for 15 min. Cells were then washed three times with PBS, and blocked for 1 h at room temperature with 5% (w/v) bovine serum albumin (BSA), Fraction V (Millipore), 0.1% Triton X-100 (Sigma) in PBS. Primary antibodies were incubated overnight at 4°C. Cells were then washed three times in PBS, and incubated with secondary antibodies conjugated to fluorophores for 1 h at room temperature. Nuclei were labeled with Hoechst (Thermo Scientific) and neutral lipids were stained with the LipidTOX Red or LipidTOX Deep Red (Thermo Scientific) fluorescent dye for 30 min at room temperature. Antibodies and dyes with working dilutions are listed in Table 1.
Lipidtox deep red
Manufactured by Thermo Fisher Scientific
Sourced in United States
LipidTOX Deep Red is a fluorescent stain used for the detection and visualization of lipid droplets in cells. It is a useful tool for studies involving lipid metabolism and cellular processes related to lipid accumulation.
Automatically generated - may contain errors
Lab products found in correlation
Bodipy 493 503, by Thermo Fisher Scientific (4 mentions)
Lipidtox green, by Thermo Fisher Scientific (2 mentions)
Rnaimax, by Thermo Fisher Scientific (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Lysotracker deep red, by Thermo Fisher Scientific (2 mentions)
C10590, by Thermo Fisher Scientific (2 mentions)
Actin rfp, by Thermo Fisher Scientific (2 mentions)
Rhodamine 800, by Merck Group (2 mentions)
Syto60, by Thermo Fisher Scientific (2 mentions)
Nucblue live readyprobes reagent, by Thermo Fisher Scientific (2 mentions)
Oleic acid, by Merck Group (2 mentions)
Bodipy 558 568 c12, by Thermo Fisher Scientific (2 mentions)
Chloroquine, by MP Biomedicals (2 mentions)
Bafilomycin a1, by Merck Group (2 mentions)
Bafilomycin, by Merck Group (2 mentions)
Vectashield h 1000, by Vector Laboratories (2 mentions)
Rat anti cd31 monoclonal antibody, by Bio-Rad (2 mentions)
Alexa fluor 633 conjugated goat anti rat igg, by Thermo Fisher Scientific (2 mentions)
Facsaria iiiu, by BD (2 mentions)
Hoechst, by Merck Group (2 mentions)
29 protocols using lipidtox deep red
1
Immunofluorescence Staining Protocol
2
Live-cell Imaging of Lipid Droplets
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Hep3B and U2OS cells were cultured at a 37 °C with 5% CO2 and in growth media consisting of MEM GlutaMax or Dulbecco’s modified Eagle’s medium (DMEM) GlutaMax supplemented with 10% fetal bovine serum (FBS) and antibiotic-antimycotic (Thermo Fisher Scientific), respectively. Two days prior to live-cell imaging experiments, 50,000 cells were seeded on 3.5 cm imaging dishes and grown to 40% confluency prior to transfection with the indicated plasmids using FuGENE HD (Promega). In some cases, cells were also incubated for 2 days before imaging with a siRNA mixture, consisting of 5 μl of RNAiMax (Thermo Fisher Scientific) and 2.5 pmol of the specified siRNA. Cells were induced to form LDs by supplementing the growth media with 200 μM OA (dissolved in ethanol) for 4 or 24 h (as indicated), before exchanging the media with either normal growth media or EBSS for 1, 4, or 18 h (as indicated) prior to imaging. LDs were identified by incubating with the LD-specific dyes Bodipy-C12-568 (Thermo Fisher Scientific), LipidTox Green (Thermo Fisher Scientific), or LipidTox Deep Red (Thermo Fisher Scientific) at volume ration of 1:10,000 for 30 min prior to imaging. In some cases, cells were treated with 1 μM Wortmannin (Cayman Chemical Company) for 30 min prior to imaging.
3
Fluorescent Labeling of Cell Nuclei
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Cell nuclei were fluorescently labelled using DAPI (Sigma Aldrich) or Hoechst 33342 (ThermoFisher). Lysotracker Green DND-26, CCF4-AM and LipidTox Deep Red were obtained from ThermoFisher.
4
Cryosectioning and RNA in situ Hybridization of hiPSC-EBs and Heart Tissue
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Cryosections of hiPSC-EBs and human heart tissues were prepared by following the sample preparation protocol for fixed frozen tissues from ACD Inc. Briefly, the tissue sections were washed with PBS, baked at 60 °C for 30 min, post-fixed with 4% PFA for 15 min at 4 °C, and then dehydrated with 50%, 70% and 100% EtOH for 1 × 5 min at room temperature. Tissues were baked in target retrieval solution at 98–102 °C for target retrieval, and then treated with Protease IV for 30 min at 40 °C. The LIPTER specific probes labelled with C2 and negative control probes were synthesized at ACD Inc. The prepared tissue and EB sections were hybridized with LIPTER or control probes following the RNAscope Fluorescent Multiplex Assay protocol. Briefly, the sections were incubated with Amp 1-FL for 30 min, Amp 2-FL for 15 min, Amp 3-FL for 1 × 30 min and Amp4 AltB-FL for 15 min at 40 °C, and washed with Wash Buffer twice at room temperature between each inculcation. For FISH and lipid double stainings, after FISH, the sections were stained with 1:1,000 LipidTOX Deep Red (ThermoFisher, H34477 ) in PBS for 30 min at room temperature. After three washes with PBS, samples were mounted with DAPI Fluoromount-G (SouthernBiotech, 0100-20) for imaging.
5
Isolation and Imaging of C. elegans Lipid Droplets
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LDs isolation from C. elegans was performed using a published method78 (link). In brief, around 100,000 synchronized L4 larval animals were washed and resuspended in buffer A (20 mM Tricine, 250 mM sucrose, pH 7.8, 0.5 mM PMSF). The worm resuspension was dounced on ice in a 15 mL Tenbroeck tissue grinders (Pyrex) for 60 initial times and subsequently transferred to a 15 mL Potter-Elvehjem tissue grinder (Wheaton) for douncing 80 more times. Worms debris and post-nuclear supernatant (PNS) was removed by centrifuge at 1000 × g for 15 min. The resulted supernatant was overlaid with buffer B (20 mM HEPES, 100 mM KCl, 2 mM MgCl2, pH 7.4) at equal ratio and used for gradient-ultracentrifugation in SW41Ti swinging bucket rotor (Beckman-Coulter). LDs were collected at buoyant fractions after a 2-h centrifugation at 10,000 × g.
For fluorescence imaging, 10 μl LD sample was diluted with 90 μl buffer B and incubated with 1:1000 diluted LipidTox Deep Red (Thermo Fisher, #H34477) for 30 min on ice. Fluorescence images were acquired using a confocal microscope (Olympus, FV1000).
For fluorescence imaging, 10 μl LD sample was diluted with 90 μl buffer B and incubated with 1:1000 diluted LipidTox Deep Red (Thermo Fisher, #H34477) for 30 min on ice. Fluorescence images were acquired using a confocal microscope (Olympus, FV1000).
6
Live-cell Imaging of HCV Infection
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Cells after electroporation with HCV RNA were seeded on 35 mm glass bottom imaging dishes (MatTek Corporation). At 48 h pe, cells were washed and cultured in phenol red-free DMEM at 37°C and 5% CO2 in the humidified incubation chamber of the imaging system. Live-cell confocal imaging was performed with the spinning disc confocal microscope PerkinElmer UltraVIEW Vox Spinning Disc CSU-X1 equipped with Nikon TiE, the EM-CCD Hamamatsu ImageEM X2 camera, and an automated Nikon perfect focus system as previously described [29 (link)]. The lipid droplets were stained with LipidTox Deep Red (Thermofisher, catalog number H34477).
7
Immunohistochemistry of Bone Sections
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Isolated long bones were fixed in 4% paraformaldehyde, decalcified using 0.5 M EDTA in phospate buffered saline (PBS) then vibratome (100‐200 μm) or frozen (20‐25 μm) sectioned. Immunofluorescence was performed using chicken or rabbit GFP antibody (Abcam, 1:1000), rabbit osterix antibody (Abcam, 1:600), rat CD31 antibody (Pharmingen, 1:200), rat CD45 antibody (Pharmingen, 1:100), rabbit perilipin antibody (Sigma, 5 μg/mL), rabbit mTert antibody (Millipore, 1:150) or rabbit osteocalcin antibody (Abcam, 10 μg/mL) and Alexa Fluor 488, 594, 633, or 647 secondary antibodies (Molecular Probes, 1:400). LipidTOX Deep Red (ThermoFisher, 1:200) was used for lipid staining. Nuclei were stained using DAPI. Sections were imaged using either a 90i Eclipse (Nikon) or LSM 700 laser scanning confocal (Zeiss) microscope.
8
Adipose Tissue Imaging Protocol
9
Oleic Acid-Induced Lipid Droplet Visualization
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Wherever relevant, cells were exposed for 1 h to 350 μM oleic acid (OA) coupled to bovine serum albumin (BSA) (0.2% w/v) to induce neutral lipids’ synthesis. LipidTox DeepRed (Thermo Fischer), was used to visualize lipid droplets or membranes enriched in neutral lipids.
10
Fixation and Staining of Cuticles and Tumours
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Cuticles and tumours were dissected on Sylgard plates in 1X PBS, and subsequently fixed on the plate in 4% formaldehyde (Polysciences, Inc., Oak Ridge, TN, USA) for 30–40 min. After fixation, tissues were transferred to a nine-well glass dissection plate for three wash steps of 15 min, on an orbital shaker at 80 rpm.
Cuticles were washed and stained in 0.05% Saponin in PBS (PBSS), while tumours were washed and stained in PBS containing 0.1% Triton X-100 (PBST). Cuticles were stained overnight at 4 °C with DAPI, Phalloidin-488 (Invitrogen, Waltham, MA, USA) at 1:100, and LipidTOX Deep Red (Thermofisher Scientific, Waltham, MA, USA) at 1:500. Cuticles were washed 3 times in PBSS before mounting on glass slides in Vectashield mounting media without DAPI (Vector Laboratories, Inc., San Francisco, CA, USA). Tumours were stained overnight at 4 °C with DAPI, washed three times in PBST and mounted on glass slides with a spacer in Vectashield mounting media without DAPI.
Cuticles were washed and stained in 0.05% Saponin in PBS (PBSS), while tumours were washed and stained in PBS containing 0.1% Triton X-100 (PBST). Cuticles were stained overnight at 4 °C with DAPI, Phalloidin-488 (Invitrogen, Waltham, MA, USA) at 1:100, and LipidTOX Deep Red (Thermofisher Scientific, Waltham, MA, USA) at 1:500. Cuticles were washed 3 times in PBSS before mounting on glass slides in Vectashield mounting media without DAPI (Vector Laboratories, Inc., San Francisco, CA, USA). Tumours were stained overnight at 4 °C with DAPI, washed three times in PBST and mounted on glass slides with a spacer in Vectashield mounting media without DAPI.
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