The tissue distribution of VSTM2A was performed on mouse tissues isolated from either 12-week-old mice or pups sacrificed 4 days postnatal (P4). To study VSTM2A expression during development, eWAT and sWAT were collected from mice at embryonic day 16.5 (E16.5) or P1, P4, P7, P14, P28, and P56. Subcutaneous WAT was collected from the inguinal region, whereas eWAT was collected directly under the testis. Tissues were either frozen for western blotting or RT-qPCR analyses or put into PBS and stained 15 min at 37°C with LipidTOX red neutral lipid stain (1:250) (Life Technologies H34476). Pictures were taken using the Evos fluorescence microscopy system (Life Technologies).
Lipidtox red neutral lipid stain
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
LipidTOX Red Neutral Lipid Stain is a fluorescent dye used to detect neutral lipids in cells. It selectively binds to neutral lipids, such as triglycerides and cholesterol esters, and emits red fluorescence upon excitation. The stain can be used in a variety of cell types to visualize and quantify neutral lipid content.
Automatically generated - may contain errors
Lab products found in correlation
H34476, by Thermo Fisher Scientific (2 mentions)
Evos fluorescence microscopy system, by Thermo Fisher Scientific (2 mentions)
Lsm800 confocal microscope, by Zeiss (2 mentions)
Mitotracker green fm, by Thermo Fisher Scientific (2 mentions)
Zen 2, by Zeiss (2 mentions)
Hoechst 33342, by Merck Group (2 mentions)
Nile red, by Merck Group (1 mentions)
Deltavision elite, by Cytiva (1 mentions)
Lsm 510, by Zeiss (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Fluorescently labeled secondary antibody, by Thermo Fisher Scientific (1 mentions)
Normal goat serum (ngs), by Merck Group (1 mentions)
Sp5 fluorescence microscope, by Leica (1 mentions)
Anti lc3, by Nanotools (1 mentions)
Anti myc, by Cell Signaling Technology (1 mentions)
7 protocols using lipidtox red neutral lipid stain
1
Spatiotemporal Analysis of VSTM2A in Adipose Tissue
2
Spatiotemporal Analysis of VSTM2A in Adipose Tissue
3
Quantifying Cellular Lipid Profiles
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Cellular cholesterol was labelled by washing cells three times in PBS followed by an incubation in PBS containing 5 μM 25-NBD-cholesterol (Avanti Polar) (10 mM stock made in 100 % ethanol) at 37 °C for 1 h. For the neutral lipid labelling cells were washed three times with PBS before being fixed with 4 % formaldehyde in PBS at RT for 20 min. The cells were then washed three times with PBS and incubated with PBS containing 1 × LipidTOX Red Neutral Lipid Stain (Invitrogen) at RT for 30 min.
Polar lipids were labelled washing the cells three times in PBS and incubating them in a 1 μg/mL Nile Red (Sigma) solution in PBS (1 mg/ml stock in 100 % acetone) at 37 °C for 1 h.
With the exception of the cholesterol labelled cells, which were imaged with a filter set for Alex Fluor 488 dye (green), all labelled cells were imaged with a filter set for Alex Fluor 594 dye (red) on a deconvolution microscope (DeltaVision Elite, Applied Precision). All images were recorded at the same setting and exposure time. Images were deconvolved using the softWoRx acquisition software (version 5.0) and then processed with ImageJ 1.43u software (NIH, USA).
Polar lipids were labelled washing the cells three times in PBS and incubating them in a 1 μg/mL Nile Red (Sigma) solution in PBS (1 mg/ml stock in 100 % acetone) at 37 °C for 1 h.
With the exception of the cholesterol labelled cells, which were imaged with a filter set for Alex Fluor 488 dye (green), all labelled cells were imaged with a filter set for Alex Fluor 594 dye (red) on a deconvolution microscope (DeltaVision Elite, Applied Precision). All images were recorded at the same setting and exposure time. Images were deconvolved using the softWoRx acquisition software (version 5.0) and then processed with ImageJ 1.43u software (NIH, USA).
4
Multimodal Imaging of Thymocytes and Lipid Droplets
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Thymocytes from B6 E15 thymic lobes cultured for 12 days were isolated and stained for surface markers and then Mitotracker Green or TMRE (as described above). All cells were subsequently analyzed on an ImageStream™ Mark II imaging flow cytometer using INSPIRE acquisition software (Amnis); 30k events were saved from samples and 1k positive events from compensation single color controls. Analysis was performed using IDEAS® version 6.2.
For lipid droplet quantification, γδ T cells expanded in vitro were stained with LipidTOX red neutral lipid stain (Invitrogen) and Hoechst 33342 (Sigma-Aldrich). Mitotracker Green FM (Invitrogen) was used to identify mitochondria. Cells were mounted onto poly-L-lysine coated slides. Images were obtained with a Zeiss LSM 800 confocal microscope using Zen 2.3 software (Zeiss) and analyzed using ImageJ.
For lipid droplet quantification, γδ T cells expanded in vitro were stained with LipidTOX red neutral lipid stain (Invitrogen) and Hoechst 33342 (Sigma-Aldrich). Mitotracker Green FM (Invitrogen) was used to identify mitochondria. Cells were mounted onto poly-L-lysine coated slides. Images were obtained with a Zeiss LSM 800 confocal microscope using Zen 2.3 software (Zeiss) and analyzed using ImageJ.
5
Intracellular Lipid Visualization of BCG-Infected Cells
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Cells were infected with BCG-GFP/IFNγ overnight in antibiotic free media supplemented with 2% FCS (to increase opsonisation and as a lipid source). Cells were washed and fixed with 4% PFA for 15 min. Cells were then washed and incubated in PBS containing LipidTOX Red Neutral Lipid Stain (Invitrogen, UK) at 1:500 dilution for a minimum of 30 min. Nuclei were costained using 1 μg/ml DAPI (Merck, UK) and cells were imaged in glass cover-slip bottomed dishes without mounting using an inverted confocal microscope. (LSM510, Carl Zeiss, Germany).
6
Multimodal Imaging of Thymocytes and Lipid Droplets
7
Indirect Immunofluorescence Staining Protocol
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Cells were fixed with 4% paraformaldehyde and processed for indirect immunofluorescence staining. Fixed cells were then permeabilized and blocked by incubating in PBS containing 0.5% Triton X-100 and 2% Normal Goat Serum (Sigma Aldrich) for 15 min at room temperature. After washing with PBS containing 0.1% Triton X-100 and 0.2% BSA, cells were conducted with primary antibodies as indicated following incubation with fluorescently labeled secondary antibodies (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). Primary antibodies used in this study are as follows: anti-PDI, GM130 (BD Biosciences, Franklin Lakes, NJ, USA), mouse and rabbit anti-Flag (Sigma Aldrich), anti-Myc (Cell Signaling), anti-LC3 (Nanotools, Teningen, Germany), anti-NS1.2 (this study), anti-NTPAse, and anti-NS4 [33 (link)]. LipidTox red neutral lipid stain (Invitrogen) was used to stain lipid droplets in fixed cells. Images were captured using a Leica SP5 fluorescence microscope. The representative images in different fields were chosen and captured from more than 100 cells. The images shown are representative of experiments carried out at least three times.
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