Time-lapse microscopy was performed using a Carl Zeiss microscope essentially as previously described (Daley et al., 2009 (link)). Bright-field images were captured every 5 min for 24 h using a Zeiss Cell Observer Z1 fitted with an environmental chamber using AxioVision software (SE64 release 4.9.1; Carl Zeiss) at 5× (Plan Neo, NA 0.16) or 20× (Plan Neo, NA 0.5) magnification. For cleft measurements, TIFF images were imported into MetaVue (version 7.7.5.0; Molecular Devices). Morphometric measurements were acquired using the line tool from calibrated images of 12 clefts from six glands grown under each growth condition.
Plan neo
Manufactured by Zeiss
The Zeiss Plan Neo is a high-precision optical microscope designed for laboratory use. It features a plan-achromatic objective lens system that provides a flat, distortion-free image across the entire field of view. The Plan Neo offers a range of magnification options and delivers reliable, consistent performance for a variety of scientific applications.
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2 protocols using plan neo
1
Time-lapse Microscopy of Organ Cleft Formation
2
Measuring Cell Migration and Polarization
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Migration was measured using a Boyden chamber. Cells were serum starved and allowed to migrate towards 10 ng/mL PDGF for 3 h. Migrated cells were stained with DAPI. Four random fields were visualized using Plan-Neo 20 × 0.5 NA in a Zeiss Axioskop2 wide-field microscope and quantified with ImageJ.
WT and Nox1y/- MEFs were seeded in 2 well silicone inserts (Ibidi) for the wound healing assays and let grow to confluency overnight. Cells were serum starved for 2 h, and the external part of the dish was filled with starvation media containing 10 ng/mL PDGF-BB. Then, the silicone inserts were carefully removed, allowing the cells to migrate to the empty area for 30 min. In some experiments, cells were fixed and processed for immunofluorescence, as described above. The staining was performed using γ-Tubulin (Sigma) as a marker of MTOC and GM130 (ECM Bioscience) as a marker of Golgi. Cells were considered polarized when Golgi or MTOC were positioned within an angle of 120° in front of the nucleus facing the wound area and as not polarized when the signal was behind this angle or on top of the nucleus.
WT and Nox1y/- MEFs were seeded in 2 well silicone inserts (Ibidi) for the wound healing assays and let grow to confluency overnight. Cells were serum starved for 2 h, and the external part of the dish was filled with starvation media containing 10 ng/mL PDGF-BB. Then, the silicone inserts were carefully removed, allowing the cells to migrate to the empty area for 30 min. In some experiments, cells were fixed and processed for immunofluorescence, as described above. The staining was performed using γ-Tubulin (Sigma) as a marker of MTOC and GM130 (ECM Bioscience) as a marker of Golgi. Cells were considered polarized when Golgi or MTOC were positioned within an angle of 120° in front of the nucleus facing the wound area and as not polarized when the signal was behind this angle or on top of the nucleus.
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