Images were collected on an inverted laser scanning confocal microscope (LSM5 Pascal, Carl Zeiss, Thornwood, NY) using either a Plan-Neofluar 10X/0.3, Plan-Neofluar 40X/0.75 or C-Apochromat 40X/1.2W objective (Carl Zeiss, Thornwood, NY). The GapYFP was excited with the 488 nm laser line using the FITC filter and all other imaging parameters were as described in (36 ). Images were collected, processed and analyzed using AIM software (Carl Zeiss, Thornwood, NY).
C apochromat 40x 1.2w objective
Manufactured by Zeiss
The C-Apochromat 40X/1.2W objective is a high-performance microscope objective designed by Zeiss. It features a numerical aperture of 1.2 and a magnification of 40X, making it suitable for various microscopy applications. The objective is designed to provide high-resolution, low-distortion imaging with excellent chromatic correction.
Automatically generated - may contain errors
4 protocols using c apochromat 40x 1.2w objective
1
Confocal Imaging of GapYFP in Cells
2
Time-lapse Confocal Imaging of Embryo Explants
3
Apoptosis Quantification in Spinal Cord
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The fraction of apoptotic cells was determined by counting active caspase-3+ cells within the regenerating portion of control and Vangl2-electroporated spinal cords. For that, non-consecutive cross-sections spanning the entire regenerate were immunostained for active caspase-3, to detect apoptotic cells, and Tuj1, to delineate the spinal cord tissue. Hoechst was used to label nuclei. Single optical sections were taken with a Zeiss C-Apochromat 40x 1.2W objective on a Zeiss LSM 780. The number of active caspase-3+ nuclei and the number of nuclei per cross-section were counted to calculate the apoptotic index as active caspase 3+/Hoechst+ for each replicate.
4
Quantifying Spinal Cord Mitotic Cleavage Planes
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Whole-mount Vangl2-overexpressing and control spinal cords at day 4 were immunostained for PH3, to identify mitotic cells, and GFAP, to visualize the apical surface of the spinal cord. Hoechst was used to label DNA. Images were taken with a Zeiss C-Apochromat 40x 1.2W objective on a Zeiss LSM 780. Late anaphase or telophase cells, with a set cleavage plane (Adams, 1996 (link)), were considered for analysis. To accurately estimate cleavage planes, only sister chromatids that appear of similar size and shape in at least 3 consecutive optical sections, and thus were perpendicular to the plane (z) of scanning, were considered further for analysis. A line through the spindle poles, inferred from the lack of DNA staining in the sister chromatids, was drawn as the mitotic spindle. The orientation of cleavage plane was determined by measuring the angle between the line orthogonal to the mitotic spindle and the apical surface of the spinal cord with the angle tool in Fiji.
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