HT1080 cells were seeded onto coverslips, grown to 50% confluency, and incubated at 37°C for 4h with inhibitor or vehicle in serum free MEM. Cells were fixed with formalin and blocked with 20% FBS in 0.05% Saponin-PBS (Saponin, EMD Millipore). Cells were incubated in 0.05% Saponin-PBS with a LBPA primary antibody (1:100) and NPC1 primary antibody (1:70), followed by an AF555 (1:400) and DY650 secondary antibody (1:400). Cells were stained with Hoechst and then mounted with PermaFluor Aqueous mounting medium (ThermoFisher). Imaging was performed by confocal microscopy (LSM800 AxioObserver Z1, Zeiss) using a 63x / 1.4NA oil Plan Apochromat objective. An average of twenty z-stacks were acquired per image, with a pixel size of 0.1 μm. Image analysis was performed using Imaris software v. 9.6.0 (Bitplane). In brief, cells were modeled as surfaces and masks were created to separate each cell into a channel. Intensity based thresholds for the NPC1 and LBPA channels were determined manually (and kept consistent for each experiment across conditions) and Pearsons’s coefficient was calculated for each cell using the colocalization module.
Lsm800 axioobserver z1
Manufactured by Zeiss
Sourced in Germany
The LSM800 AxioObserver Z1 is a laser scanning confocal microscope manufactured by Zeiss. It is designed for high-resolution imaging of biological samples. The microscope features a fully motorized stand with a built-in autofocus system and a range of objectives for versatile imaging applications.
Automatically generated - may contain errors
Lab products found in correlation
Zen 2, by Zeiss (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Permafluor aqueous mounting medium, by Thermo Fisher Scientific (1 mentions)
Saponin, by Merck Group (1 mentions)
Celltracker blue cmac dye, by Thermo Fisher Scientific (1 mentions)
Dextran fluorescein, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Pi rnase solution, by BD (1 mentions)
Sca1 apc, by Miltenyi Biotec (1 mentions)
Fitc phalloidin, by Merck Group (1 mentions)
8 protocols using lsm800 axioobserver z1
1
Quantitative Analysis of NPC1 and LBPA Colocalization
2
Dextran Uptake Assay in Vero Cells
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Vero cells were seeded onto coverslips and grown to approximately 60% confluency. Cells were pre-treated for 1 h with inhibitor or vehicle in serum-free MEM, followed by a 30 min incubation with Dextran-Fluorescein (10,000 MW, Anionic, 1 mg/mL, ThermoFisher) and CellTracker Blue CMAC Dye (ThermoFisher). Cells were washed, fixed, and imaged by confocal microscopy (LSM800 AxioObserver Z1, Zeiss, Oberkochen, Germany). Image analysis was performed using Imaris image analysis software (Bitplane, Zurich, Switzerland) whereby dextran containing vesicles were modeled as spots and counted for each cell.
3
Fixation and Fluorescent Imaging of E. coli
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E. coli cells were fixed with 4% formaldehyde at 37 °C, 220 r.p.m. for 10 min. After fixation, cells were collected by centrifugation at 12,000 r.p.m. for 1 min. Cell pellets were washed twice with PBS and then resuspended in PBS containing Hoechst33342. The cells were then incubated at room temperature for 1 hr. Add 2.5 μL of cell solution and 1 μL of pre-melted 1.2% low melting-point agar was mixed on the glass slide and covered with a coverslip for observation. Images were captured under Plan-Apochromat 63×/1.40 Oil DIC M27 objective on a Carl Zeiss LSM 800 (Axio Observer Z1) inverted fluorescence confocal microscope.
4
Immunofluorescence Imaging of Cell Nuclei
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Immunofluorescence of cell nuclei was performed using a micromanipulator system (Fig. S5 ). Individual nucleus-like structures were fixed with 3.7% paraformaldehyde (w/v) in phosphate buffered saline (PBS) for 15 min and permeabilised with 2% Triton X-100 in PBS for 2 h at room temperature. After fixation and permeabilisation, the samples were incubated in the blocking buffer (3% BSA + 0.02% Tween in PBS) for 1 h at room temperature and incubated with primary antibodies against histone H3 (1:50) and lamin B2 (1:50) in blocking buffer at 4 °C overnight. The samples were washed with blocking buffer for 15 min at room temperature; this step was performed three times. The samples were then incubated with secondary antibodies (1:200) for 1 h at room temperature. The DNA was stained with 0.05 µg/ml of 4′, 6-diamidino-2-phenylindole (Thermo Fisher Scientific., D3571) in blocking buffer for 1 min. Finally, the samples were mounted on a slide glass and examined with a laser-scanning confocal microscope (Zeiss LSM800 Axio Observer Z1) and imaging software (Zeiss ZEN 2).
5
Imaging Nanoparticle Uptake in Macrophages
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BMDM were treated with Cy5.5-conjugated NPs for 1.5 h then washed twice with PBS. 1% paraformaldehyde in PBS was added to the cells for fixation for 15 min at room temperature. The cells were then washed and simultaneously blocked and permeabilized with a 1% BSA and 0.1% Triton X-100 PBS solution for 30 min at room temperature. Cell were then stained with a LAMP1-eF450 (1:100; Ex/Em: 405 nm/450 nm; eBioscience) primary antibody in PBS for 30 min. BMDM were washed again and immediately prior to mounting were stained with 0.5 mL/million cells of PI-RNAse solution (Ex/Em: 493 nm/636 nm; BD). BMDM were imaged on the Zeiss LSM800 AxioObserver Z1. Images were later analyzed using ImageJ.
6
Visualizing Intracellular Cholesterol in HT1080 Cells
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HT1080 cells were seeded onto coverslips, grown to 50% confluency, and incubated at 37°C for 4h with inhibitor or vehicle in serum free MEM. Cells were fixed with formalin, incubated with Filipin III (50 μg/mL in PBS) for 2h at RT, and coverslips mounted with PermaFluor Aqueous mounting medium. Cells were imaged by confocal microscopy (LSM800 AxioObserver Z1, Zeiss) using a 63x / 1.4NA oil Plan Apochromat objective. An average of twenty z-stacks were acquired per image, with a pixel size of 0.1 μm.
7
Lipid and Stemness Profiling of Hematopoietic Stem Cells
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BM from S. typhimurium (72 hours) infected C57BL/6 J, CD36+/+, and CD36−/− animals were lineage depleted and CD117 enriched (LK). These LK cells were then stained with 5 μM Hoechst 33342 (ThermoFisher, Waltham, MA, USA) and a fluorescent neutral lipid dye 4- difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY 493/503) (1 μM) (ThermoFisher, Waltham, MA, USA) at room temperature for 30 minutes. The cells were washed twice in 1× PBS by centrifugation at 400 × g for 5 minutes and stained with the cell surface antibody Sca1-APC (Miltenyi Biotec, Bergisch Gladbach, Germany) for 20 minutes in the dark. The LK cells positive for Sca1 marker were then termed LSKs. The cell suspension was washed again with 1× PBS and resuspended in 200 μL of FluoroBrite DMEM medium supplemented with 10% fetal calf serum (FCS) and plated in a black-walled imaging plate. Confocal images were acquired on a Zeiss LSM 800 Axio Observer.Z1 using a ×63 water objective and data were collected on Zenbio. Fiji imageJ 2.0.0 software was used for image processing.
8
Immunofluorescence Staining Protocol for Cultured Cells
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Cells were cultured for 48 h on glass coverslips, then washed with PBS, fixed with 4% paraformaldehyde for 15 min at room temperature, permeabilized with 0.25% TritonX-100 (10 min), and non-specific sites were blocked with 2% BSA in TBST for 1 h. Slides were then incubated with the specific primary antibody suspended in 2% BSA in TBST (Table 2 ) overnight at 4 °C. After washing several times with TBST, cells were incubated with the corresponding secondary antibody labeled with Alexa Fluor 594 diluted in 2% BSA in TBST for 1 hour in the dark. To visualize actin filaments, cells were incubated with Phalloidin-FITC (Sigma-Aldrich, St. Louis, MO, USA) and DAPI (4′,6-diamidino-2-phenylindole) for nuclei staining. Finally, cells were observed in a laser scanning confocal microscope LSM 800, AxioObserver Z.1 (Zeiss, Oberkochen, Germany) using ZEN 2.6 software (Zeiss).
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