As previously reported [7 (link)], we stained and visualized whole-mount adipose tissue. Mice were sacrificed by cervical dislocation, after which the VAT was removed using sterile technique and minced into small pieces (~2–3 mm) using a scalpel. We washed the tissue pieces, which were then fixed in cellFIX (Cat. 340181, BD) for 60 min and permeabilized with 0.1% Triton X-100 for 10 min. The specimens were then blocked with 5% bovine serum albumin and incubated first with a primary antibody [F4/80 (BM-8, eBioscience)] overnight at 4°C and then with Alexa Fluor 488-conjugated secondary antibody (Molecular Probes) for 1 h. The tissues were counterstained for 1 h with BODIPY 558/568 (Molecular Probes) to visualize adipocytes and with DAPI (Molecular Probes) to visualize nuclei. We excited the tissue samples using four laser lines (405 nm, 488 nm, 568 nm, and 800 nm) and collected the emission through appropriate narrow band-pass filters in a confocal microscope (LSM 710, Carl Zeiss). The images were acquired and processed by LSM 710 software.
Bodipy 558 568
Manufactured by Thermo Fisher Scientific
Sourced in United States
BODIPY 558/568 is a fluorescent dye with absorption and emission maxima at 558 nm and 568 nm, respectively. It can be used as a fluorescent label for biomolecules such as proteins, nucleic acids, and lipids.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710, by Zeiss (6 mentions)
Bodipy 493 503, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 488 conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
F4 80 bm8, by Thermo Fisher Scientific (2 mentions)
Cellfix, by BD (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Fluorescent mounting medium, by Agilent Technologies (2 mentions)
Fm4 64fx, by Thermo Fisher Scientific (2 mentions)
Vectashield, by Vector Laboratories (2 mentions)
Fv1000, by Olympus (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Rat anti mouse f4 80, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Thermo Fisher Scientific (1 mentions)
Dulbecco s phosphate buffered saline pbs, by Merck Group (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Tcs sp2, by Leica (1 mentions)
Doxycycline, by Merck Group (1 mentions)
Puromycin, by Thermo Fisher Scientific (1 mentions)
Bodipy fl c16, by Thermo Fisher Scientific (1 mentions)
Bodipy 558 568 c12, by Thermo Fisher Scientific (1 mentions)
14 protocols using bodipy 558 568
1
Whole-Mount Adipose Tissue Imaging Protocol
2
Adipose Tissue Immunostaining Protocol
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All mice were euthanized at 68 to 70 weeks of age for histological and immunological analyses. As previously reported [18 (link)], we stained and examined whole mounts of adipose tissue. Mice were sacrificed by cervical dislocation, after which the VAT was removed by a sterile technique and minced into small pieces (~2–3 mm) with a scalpel. The tissue pieces were washed, fixed in cellFIX (Cat. 340181, BD) for 60 min, and permeabilized with 0.1% Triton X-100 for 10 min. Then the specimens were blocked with 5% bovine serum albumin, incubated with the primary antibody [F4/80 (BM-8, eBioscience, San Diego, CA, USA)] overnight at 4°C, and then incubated with the Alexa Fluor 488-conjugated secondary antibody (Molecular Probes, Eugene, OR, USA) for 1 h. The tissues were counterstained for 1 h with BODIPY 558/568 (Molecular Probes) to visualize adipocytes and with 4',6-diamidino-2-phenylindole (DAPI; Molecular Probes) to visualize nuclei. For confocal microscopy (LSM 710, Carl Zeiss, Jena, Germany), the tissue samples were illuminated with four laser lines (405 nm, 488 nm, 568 nm, and 800 nm) and emissions were collected through appropriate narrow band-pass filters, after which images were acquired and processed by LSM 710 software.
3
Visualization of Adipose Tissue In Vivo
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Visualization of nonfixed living adipose tissue was performed according to a previously described procedure 15 . Briefly, samples that were harvested at 2 and 12 weeks in vivo were cut into 3‐ to 5‐mm pieces within 2 hours after sampling, incubated with boron‐dipyrromethene (Bodipy) 558/568 (Molecular Probes) for 15 to 30 minutes, washed with stroke‐physiological saline solution (SPSS) 3 times, and observed under a confocal microscope system (Zeiss, LSM710, Germany). Six to 10 images were vertically acquired at an interval of 5 μm for each low‐magnification field of view.
4
Visualization of Adipose Tissue Macrophages
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Visceral WAT was formalin-fixed, cut into 2–3 mm sections, and transferred to Dulbecco’s phosphate buffered saline (PBS, Sigma Aldrich) for 48 h at 4°C. Adipose tissue was permeabilized in 1% Triton X-100 (Fisher) in PBS for 10 min before being stained with rat anti-mouse F4/80 (Invitrogen, Carlsbad) to mark macrophages and detected with donkey anti-rat IgG conjugated to Alexa Fluor488 (Invitrogen). Tissues were incubated with primary antibodies overnight at room temperature (RT) in the dark and washed 3× with staining buffer before being subjected to secondary stain for 2 h and wash (3×) in PBS. Stained samples were then counterstained for 15 min at RT with 5 μM BODIPY 558/568 (Molecular Probes, Inc.) to visualize lipid. Adipose tissue samples were placed directly on a coverslip with buffer and visualized.
5
Immunofluorescence Staining of HepG2 Cells
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After the indicated treatments, HepG2 cells were fixed with 4% paraformaldehyde for 30 min at room temperature. Cells were then treated with 0.1% Triton X-100 for 20 min and blocked with 10% goat serum for 1 h at room temperature. The primary antibodies for Fsp27β or CIEDC were added and the reaction was incubated overnight at 4 °C. Anti-rabbit IgG antibody conjugated with Alexa Fluor 488 (A11008; Molecular Probes, Eugene, OR) was used as a secondary antibody. Bodipy 558/568 (D3835; Molecular Probes) was used for lipid staining. The nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min at room temperature. The sections were observed under a Zeiss 200 M inverted microscope, and the images were collected using a confocal laser scanning microscope (TCS SP2; Leica Microsystems GmbH).
6
Detailed Antibody and Lipid Reagents
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Anti-VPS13D (A304-691A, Bethy Laboratories. Inc), Anti-GFP (AE011, Abclonal), anti-Halo (G9211; Promega), anti-Tubulin (100109-MM05T; Sinobiological), anti-actin (20536-1-AP; Proteintech), anti-VDAC1 (55259-1-AP; Proteintech), anti-TSG101 (A1692; Abclonal) were used at 1:1000 dilutions for western blots. Anti-VPS13D (A304-691A, Bethy Laboratories. Inc) and anti-TSG101 (SC7964; Santa Cruz Biotechnology) antibodies were used 1:100 for immunofluorescence. The following reagents were used in this study: Oleic acid (O1008; sigma); Palmitic acid (P0500; sigma); Doxycycline (1225984; Sigma), BODIPY 493/503 (ThermoFisher Scientific, D3922), BODIPY 558/568 (ThermoFisher Scientific, D2219), NBD-C12 (Abcam, Ab145361), BODIPY 558/568 C12 (ThermoFisher Scientific, D3835), BODIPY FL C16 (ThermoFisher Scientific, D3821), Janilia Fluo® 646 HaloTag® Ligand (GA1120; Promega) and antibiotics such as G418 (10131027) and puromycin (A1113803) were obtained from ThermoFisher Scientific. All EM reagents were purchased from Electron Microscopy Sciences. Lipids were purchased from Avanti Polar Lipids: NBD-PC (810133), NBD-PE (810144), NBD-PS (810198), NBD-PA (810138), NBD-ceramide (810211).
7
Fluorescent Lipid Droplet Imaging
8
Fluorescent Lipid Droplet Imaging
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Cells were incubated with 20 µg/ml BODIPY 558/568 or BODIPY 493/503 (ThermoFisher) for 45 min at 37°C in PBS, rinsed to remove excess stain followed by a further incubation of 60 min at 37°C in cell culture media. Cells were either viewed live or fixed with 4% paraformaldehyde at 4°C for 30 min. Fixed cells were washed with PBS prior to mounting fluorescent mounting medium (DAKO) and coverslipped.
9
Cell Labeling and Flow Cytometry
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The A549 and H1299 cells were prepared and blocked with rat IgG (10 μg/mL; Sigma) for at least 20 min on ice. After that, they were washed with staining buffer (PBS containing 2% FBS). Then, the cells were labeled with the indicated antibodies (1:100) for 30 min at 4 °C. Dead cells were excluded using a Fixable Viability Dye Efluor 780 (1:1000; Cat No. 65–0865-14, eBioscience). Bodipy558/568 (Cat No. D3835) was obtained from Thermo Fisher Scientific. Mito-Tracker Green probes (Cat No. C1048) were from Beyotime (Shanghai, China). Flow cytometry (FCM) was performed on BD FACS Canto II platforms, and the results were analyzed with FlowJo software version 10.0.7 (TreeStar). Sorting was performed on a BD FACSAria II instrument (BD Biosciences).
10
Multimodal Imaging of Neurovascular Structure
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IFP sections 300 µm thick were incubated in blocking solution (2% Normal Horse Serum and 0.2% triton X-100 in PBS) at room temperature (RT) and incubated for 24 hours at RT with BODIPY 558/568 (1:1000, D3835, Invitrogen, Carlsbad, CA, USA), tyrosine hydroxylase 488 (1:250, AB1542, Milipore, Darmstadt, Germany) and biotinylated lectin (1:100, B-1105, AbCys, Courtaboeuf, France). Then sections were incubated overnight at 4 °C with streptavidin Alexa-647 (1:100, S21374, Invitrogen, Carlsbad, CA, USA), mounted on a coverslip and imaged using a confocal laser scanning microscope (LSM780, Carl Zeiss, Oberkochen, Germany). Second harmonic generation signal allowing visualization of fibrous collagen was acquired using a mode-locked 890 nm laser and was measured at 445 nm with a 20 nm bandpass filter. Images were processed using Fiji software (NIH, Bethesda, MD, USA).
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