Cells were fixed using 4% PFA or 100% methanol (MeOH, Sigma-Aldrich) for 10 min (PFA) or 15 min (MeOH) at room temperature (PFA) or –20°C (MeOH). Following PFA fixation, the cells were permeabilized using 0.1% Triton X-100 (Sigma-Aldrich) in PBS. Cells fixed using MeOH were directly blocked using 5% fetal bovine serum in PBS before incubating with primary and secondary Alexa-labeled antibodies. The coverslips (1943-10012A, Bellco) were mounted using Mowiol on glass slides and imaged using Zeiss LSM 780 laser-scanning confocal microscope ×63/1.4 NA oil immersion objective lens or Olympus FV3000 confocal laser-scanning microscope with a ×60 Plan Apo N objective (oil, 1.42 NA). Data from three independent experiments were subjected to analysis by the automated image analysis program, Motion Tracking (Kalaidzidis, 2007 (link); Rink et al., 2005 (link); http://motiontracking.mpi-cbg.de ) as described in detail Source data 2 .
Planapo n objective
Manufactured by Olympus
The PlanApo-N objective is a high-performance microscope objective lens designed for a range of microscopy applications. It features a plan-apochromatic optical design that provides excellent image flatness and chromatic correction. The objective delivers high numerical aperture and high resolution capabilities.
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Lab products found in correlation
Fv3000 confocal laser scanning microscope, by Olympus (5 mentions)
Deltavision imaging system, by GE Healthcare (3 mentions)
Fitc conjugated phalloidin, by Merck Group (2 mentions)
Axio observer z1, by Zeiss (2 mentions)
Triton x 100, by Merck Group (1 mentions)
Lsm 780, by Zeiss (1 mentions)
Fv3000, by Olympus (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
D1306, by Merck Group (1 mentions)
F6182, by Merck Group (1 mentions)
Aria 3, by BD (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (1 mentions)
Mitotracker deep red, by Thermo Fisher Scientific (1 mentions)
Orca flash 4, by Hamamatsu Photonics (1 mentions)
Uplanfln objective, by Olympus (1 mentions)
Co2 independent media, by Thermo Fisher Scientific (1 mentions)
Deltavision core microscope, by Cytiva (1 mentions)
Uplanapo objective, by Olympus (1 mentions)
Fv1000, by Olympus (1 mentions)
14 protocols using planapo n objective
1
Immunofluorescence Imaging Protocol for Cellular Analysis
2
Immunofluorescence Staining of Cultured Cells
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Cells were plated at a seeding density of 3 × 104 on a coverslip in a 12-well plate. After treatment, the cells were washed thrice with ice-cold PBS, followed by 4% formaldehyde fixation and subsequent permeabilization with 0.1% Triton X-100 solution. Post blocking for 1hr at RT using 2% BSA solution, cells were incubated with appropriate primary antibodies overnight at 4°C. Cells were then washed thrice with PBS and incubated with Alexa-Flour 555 anti-rabbit IgG secondary antibody for 1hr at RT. Subsequently, the cells were counterstained with DAPI (Invitrogen, D1306, lot no. 1673432) and mounted using fluoroshield (Sigma, F6182, lot no. MKCN2676). For Ki67 staining, primary antibody incubation was directly followed by nuclei counterstaining using Hoechst 33342 (Invitrogen, HI399, lot no. 1932847). Imaging was performed using Olympus FV3000 confocal laser scanning microscope with a 60× Plan Apo N objective (oil, 1.42 NA), and image analysis was performed using Image J software.
3
Live-cell Imaging of Protein Colocalization
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Cells were cotransfected with GFP and mCherry-fused proteins for 12 h, followed by trypsinization, and were seeded on glass-bottom dishes coated with gelatin. Cells in L-15 complete media were incubated for 4 h at 37°C in an incubator and further imaged with an Olympus FV3000 confocal laser-scanning microscope with a 60× Plan Apo N objective (oil, 1.42 NA) on an inverted stage. Dual-color sequential imaging was performed using 488-nm and 561-nm lasers to excite GFP and mCherry-fusion proteins, respectively. Images were acquired and processed using FV31S-SW software.
4
Mitochondrial Staining and Imaging Protocol
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MitoTracker™ Deep Red (Invitrogen, M22426, lot no. 2112250) stock solution was prepared as per the manufacturer's instructions. 1 × 105 cells were seeded in a well of 12-well plate and allowed to attach for 24 h. 70 nM working solution of MitoTracker made using phenol red-free DMEM (Gibco, 21063-029, lot no. 1967601) or phenol red-free RPMI (Gibco, 11835-030, lot no. 1951096) was added to the cells. After 45 min of incubation in the dark at 37°C, the cells were washed with PBS and stained for nucleus using Hoechst 33342. Subsequently, the cells were trypsinized, washed twice with PBS, finally resuspended in PBS, and subjected to flow cytometry using fluorescence-activated cell sorting (FACS) Aria III by Becton Dickinson. Analysis of the data was performed using FlowJo software (version 10.7.1). For fluorescence imaging, MitoTracker was added to the spheroid culture at a final concentration of 150 nM, followed by incubation in the dark at 37°C. The staining was then visualized using Olympus FV3000 confocal laser scanning microscope with a 60× Plan Apo N objective (oil, 1.42 NA).
5
Hypoxia Regulates MENA Isoform Localization
6
Imaging Hypoxia-Induced Actin Dynamics
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After puromycin selection, cells were transfected with overexpression plasmids pCMV-hMENA11a and pCMV-hMENAΔ11a. Cells were seeded in 12-well plates on coverslips. After 48 h of hypoxic or normoxic treatment, cells were washed and fixed using 4% formaldehyde and permeabilized using 0.1% Triton X-100. After washing, cells were then incubated with FITC-conjugated phalloidin (Sigma, P5282) for 4 h, washed with phosphate-buffered saline (PBS) and mounted. Sections were analyzed using an Olympus FV3000 confocal laser scanning microscope with a 60× Plan Apo N objective (oil, 1.42 NA).
7
Quantifying Mitotic Spindle Dynamics
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FDAPA analysis and images used for quantifying astral length, inner spindle intensity, spindle orientation, and TACC3 and MCAK spindle localization were acquired using a Marianas (Intelligent Imaging Innovations) spinning-disk confocal system based on a microscope (Axio Observer Z1; ZEISS) equipped with a camera (ORCA-Flash 4.0; Hamamatsu Photonics). Images were taken using a 63× 1.4 NA Apochromat objective (ZEISS). Images for TACC spindle localization in U2OS cells were taken using a 100× 1.4 NA Plan-Apochromat objective (ZEISS). The images were z-projected using Slidebook software 5.5 (Intelligent Imaging Innovations). All other images were acquired using a DeltaVision imaging system (GE Healthcare) equipped with an sCMOS camera (PCO Edge 5.5). Images were taken using a 60× 1.42 NA PlanApo-N objective (Olympus) at room temperature. Serial z stacks of 0.2-µm thickness were obtained and deconvolved using SoftWoRx software 6.1.l. For live-cell imaging, media was changed to CO2-independent media (Gibco) 12 h before imaging. Live-cell image sequences were acquired at 1-min intervals for 12 h in 2-µm serial z sections using a 40× 1.42 NA UPlanFL-N objective (Olympus) at 37°C.
8
Quantification of Dynein Spindle Pole Localization
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Images were acquired on a DeltaVision Core microscope (Applied Precision) equipped with a CoolSnap HQ2 CCD camera (Photometrics). A 100×, 1.4 NA U-PlanApo objective (Olympus) was used to image fixed cells, and a 60×, 1.42 NA Plan Apo N objective (Olympus) was used for live-cell imaging. Images were deconvolved and maximally projected. The fluorescence is not scaled equivalently in each panel to clearly demonstrate the qualitative localization of each protein.
To quantify the spindle pole accumulation frequency of dynein heavy chain, each replicate included 100 cells for each condition. The percentage of mitotic cells with clear, strong foci of the dynein-GFP signal on the spindle poles was denoted. 3-4 biological replicates were analysed for each condition and the mean percentage of cells (±s.d.) with strong dynein signal for those replicates was plotted. For ZW10 and MAD1, 2 biological replicates were analysed for each condition and the mean percentage of cells (±s.d.) with spindle pole-localized signal for those replicates was plotted.
Line scans were generated through the ‘Plot profile’ function in Image J, using maximally projected, but not deconvolved, images.
To quantify the spindle pole accumulation frequency of dynein heavy chain, each replicate included 100 cells for each condition. The percentage of mitotic cells with clear, strong foci of the dynein-GFP signal on the spindle poles was denoted. 3-4 biological replicates were analysed for each condition and the mean percentage of cells (±s.d.) with strong dynein signal for those replicates was plotted. For ZW10 and MAD1, 2 biological replicates were analysed for each condition and the mean percentage of cells (±s.d.) with spindle pole-localized signal for those replicates was plotted.
Line scans were generated through the ‘Plot profile’ function in I
9
Imaging Microtubule Network in Pupal Wings
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Images were acquired at room temperature (20–22°C). The subapical domain of the cell was identified using the localization of both tubulin and E-cadherin (see Figure 3 ). Pupal wings were oriented along the proximodistal axis using v4 as a reference. For analysis of Ds-EGFP and Patronin-GFP distribution and polarity, an upright confocal microscope (FV1000; Olympus) using a 60× 1.42 NA oil PlanApoN objective lens was used. Sixteen-bit depth images were taken at a magnification of 12.8 pixels/μm with 0.15 (Ds-EGFP) or 0.38 (Pat-GFP) µm between z-sections. Images of the subapical microtubule network were acquired using a Zeiss Airyscan microscope and the 63× objective lens. Z-stacks consisted of seven sections with 23.5 pixels/μm in XY resolution and 0.185 µm distance between sequential z-sections. All processing was done at 6.5 power in ZEN software. Representative images used for figure preparation are the average projections of the region of interest/analysis. Figures were assembled using Adobe CS3 Photoshop and Illustrator (http://www.adobe.com ). The processing of images shown in the figures involved adjusting gamma settings.
10
Vimentin Filament Length Measurement
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To measure the lengths of vimentin filaments (see Supplementary Fig. 1 ), we prepared five 1.5 mL reaction tubes with 15 μL of a mix of 2.3 μM vimentin in CB including all additions as used for the TIRF experiments, such as methyl cellulose, GTP, and oxygen scavenger (see the previous section for the exact composition of the buffer). We then incubated the mix at 37 ∘C for 5, 10, 20, 30, or 45 min. The filament assembly was stopped by adding 25 volumes of buffer to the tubes. Five microliters of each diluted mix was then pipetted on a cover glass and a second cover glass was placed on top. Images were taken with an inverted microscope (IX81, Olympus) using the cellSens Dimensions software (version 1.18, Olympus), a 60× oil-immersion PlanApoN objective (Olympus), and an ORCA-Flash 4.0 camera (Hamamatsu Photonics). The filament lengths were determined using the semi-automated JFilament 2D plugin (Lehigh University, Bethlehem, PA, USA, version 1.02) for ImageJ (version 2.0.0-rc-69/1.52p).
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