Upon fixation with 4% PFA, cells were washed with HBSS and mounted in Fluoromount (Sigma Aldrich). Fluorescence microscopy was performed using the Observer Z1 microscope (Zeiss) with a 63× planApo oil immersion objective (1.40 NA). For analysis of dendritic branching, GFP images were used. Sholl analysis was performed using ImageJ.
Planapo oil immersion objective
Manufactured by Zeiss
The 63× planApo oil immersion objective from Zeiss is a high-performance microscope lens designed for advanced imaging applications. It offers a high numerical aperture and a flat field of view, providing excellent optical performance and resolution. The objective is intended for use with oil immersion microscopy techniques.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Merck Group (2 mentions)
Deltavision rt microscope, by Cytiva (2 mentions)
Observer z1 microscope, by Zeiss (1 mentions)
Fluoromount, by Merck Group (1 mentions)
Lsm 800, by Zeiss (1 mentions)
Zen 2, by Zeiss (1 mentions)
Lsm 780, by Zeiss (1 mentions)
Imspector software, by Abberior (1 mentions)
Instrument expert line sted, by Abberior (1 mentions)
Lipofectamine, by Thermo Fisher Scientific (1 mentions)
Glass bottomed dishes, by MatTek (1 mentions)
Glass bottom dishes, by MatTek (1 mentions)
6 protocols using planapo oil immersion objective
1
Dendritic Branching Analysis of GFP-labeled Cells
2
Multi-Modal Microscopy Imaging Protocol
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All micrographs in figures are representative for the respective experimental sample. Unless otherwise noted, images were acquired with a CSU-W1 Yokogawa Spinning Disk Field Scanning Confocal System coupled to a Hamamatsu ORCA-Fusion C14440 sCMOS camera using a Nikon 60× Plan Apo Lamba Oil Immersion objective [numerical aperture (NA), 1.40]. Images in Fig. 1 (A, C, and E) and figs. S1 (A, E, and G) and S6B were acquired using a Zeiss LSM 800 point scanning confocal using a Zeiss 63× Plan Apo oil immersion objective (NA, 1.40) with Airy Scan SR mode activated. Raw Airyscan images were processed using Zeiss ZEN 2.3 software.
3
Super-Resolution Microscopy and FCS Protocols
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sSTED-FCS measurements were performed on the Abberior Instrument Expert Line STED super-resolution microscope (Abberior Instruments GmbH, Göttingen, Germany) using 485- and 640-nm pulsed excitation laser sources and a pulsed STED laser operating at 775 nm and an 80-MHz repetition rate. The fluorescence excitation and collection were performed using a 100×/1.40 numerical aperture (NA) UPlanSApo oil immersion objective (Olympus Industrial, Southend-on-Sea, UK). All acquisition operations were controlled by Imspector software (Abberior Instruments GmbH), and point FCS data for the calibration of the observation spot sizes were recorded using a hardware correlator (Flex02-08D, correlator.com , operated by the company’s software).
Confocal microscope FCS measurements were performed on a Zeiss LSM 780 (Zeiss, Iena, Germany) using a HeNe 633-nm laser as the excitation source. Fluorescence excitation and collection were performed using a 63×/1.40-NA PlanApo oil immersion objective (Zeiss). Acquisition operations were controlled by the Zeiss Zen software. Point FCS was used to calibrate the observation spot with the LSM 780 internal hardware correlator.
Confocal microscope FCS measurements were performed on a Zeiss LSM 780 (Zeiss, Iena, Germany) using a HeNe 633-nm laser as the excitation source. Fluorescence excitation and collection were performed using a 63×/1.40-NA PlanApo oil immersion objective (Zeiss). Acquisition operations were controlled by the Zeiss Zen software. Point FCS was used to calibrate the observation spot with the LSM 780 internal hardware correlator.
4
Super-Resolution Imaging of HeLa Cells
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Fixed HeLa cell samples were imaged with an Elyra SR-SIM system (Zeiss) equipped with a ×100 magnification/1.46 N.A. Plan-Apo oil immersion objective (DIC); 488-, and 561-nm OPSL lasers; and PCO edge sCMOS cameras. SI image stacks were acquired with a z-distance of 125 nm and with 15 raw SI images per plane (five phases, three angles). The SI raw datasets were computationally reconstructed with channel-specific measured optical transfer functions (OTFs) and a Wiener filter set to 0.002 using the Imaris software packages (Bitplane) to obtain a super-resolution 3D image stack with a lateral (x,y) resolution of ∼120 nm and an axial (z) resolution of ∼300 nm.
5
Determining Talin Protein Localization
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Talin1 and talin2 knockout cells were generated and cultured as described in Atherton et al. (2015) (link). Transient transfections were performed using Lipofectamine and Plus reagents (Life Technologies) as per the manufacturer's instructions. Cells transfected with GFP-talin proteins were incubated overnight on glass-bottomed dishes (MatTek), fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100 (Sigma). Samples were incubated with the primary antibody for 60 min and then washed thrice with PBS. Secondary antibody staining followed the same procedure. Fixed samples were imaged using a Delta Vision RT microscope (Applied Precision) equipped with a 60×/1.42 Plan Apo oil-immersion objective (Zeiss). Images were acquired with a CoolSnap HQ camera (Photometrics). Images were background subtracted, a region of interest was selected around an individual peripheral adhesion (five per cell), and the integrated density measured for both channels. Dividing the values from paxillin or DLC1 by talin then produced a ratio.
Further details can be found inSupplemental Experimental Procedures .
Further details can be found in
6
Visualizing Protein Localization in Cells
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Cells transfected with GFP- and/or mCh-tagged proteins were incubated overnight on glass bottom dishes (MatTek), fixed with 4% paraformaldehyde and permeabilised with 0.5% Triton X-100 (Sigma). Samples were incubated with the primary antibody for 60 min, and then washed thrice with PBS. Secondary antibody staining followed the same procedure. Fixed samples were imaged using a Delta Vision RT microscope (Applied Precision) equipped with a 60 × /1.42 Plan Apo oil immersion objective (Zeiss). Images were acquired with a CoolSnap HQ camera (Photometrics).
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