Under deep anesthesia, ICR mice were fixed transcardially with 4% PFA in phosphate-buffered saline, pH 7.4. Fifty- or 100-μm-thick coronal sections were prepared with microslicer (VT1200S; Leica Biosystems). Sections were stained with Hoechst 33342 and mounted. Fluorescence images of 100-μm-thick sections were taken with a confocal laser-scanning microscope (TCS SP8; Leica Microsystems) using HC PL APO CS2 20×/0.75 NA multiple immersion lens (Leica Microsystems). Images were taken using the tile scan function of the confocal microscope. Fluorescence images of 50-μm-thick sections were taken using HC PL APO CS2 100×/1.40 NA oil immersion lens (Leica Microsystems). Basal dendrites of the cortical layer 2/3 pyramidal neurons were randomly sampled and captured. Image stacks were deconvolved using Huygens Essential version (Scientific Volume Imaging). Spines on tertiary dendrites were identified and counted in the 3D projection images with respect to each mouse. For immunohistochemical analysis, 50-μm-thick sections were prepared as described above, and immunostained with rabbit anti-HA antibody (Covance), followed by incubation with Alexa Fluor 488-conjugated anti-rabbit antibody (Invitrogen) and DAPI. Images were taken as described above.
Hc pl apo cs2 20 0.75 na multiple immersion lens
Manufactured by Leica
The HC PL APO CS2 20×/0.75 NA multiple immersion lens is an objective lens designed for use in microscopes. It has a magnification of 20x and a numerical aperture (NA) of 0.75, which enables high-resolution imaging. The lens is designed for use with various immersion media, such as air, water, or oil, to optimize performance for different imaging applications.
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3 protocols using hc pl apo cs2 20 0.75 na multiple immersion lens
1
Imaging Dendritic Spines in Cortical Neurons
2
Quantitative Analysis of Neuronal Morphology
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Images of culture experiments were taken with a confocal laser-scanning microscope (TCS SP8; Leica Microsystems) using HC PL APO CS2 20×/0.75 NA multiple immersion lens (Leica Microsystems) or HC PL APO CS 10×/0.40 NA multiple immersion lens (Leica Microsystems) under constant conditions in terms of laser power, pinhole size, gain, z-steps, and zoom setting throughout the experiments. All quantitative measurements were performed with ImageJ 1.52a software [42] . For the quanti cation of MAP2 and tau staining areas, z-series of optical sections were projected by the brightest point method.
MAP2 and tau staining areas were de ned as area in which staining signal intensites were 2 and 3 times stronger than those of background signals on the same eld, respectively. For analysis of the time course of neurite outgrowth, z-series of optical sections were projected by the brightest point method. Neurite of cortical neurons was identifed by Tuj1 signals, and the length of the longest neurite, the number of neurites emerging from the soma, and the numbers of branches per longest neurite were measured.
MAP2 and tau staining areas were de ned as area in which staining signal intensites were 2 and 3 times stronger than those of background signals on the same eld, respectively. For analysis of the time course of neurite outgrowth, z-series of optical sections were projected by the brightest point method. Neurite of cortical neurons was identifed by Tuj1 signals, and the length of the longest neurite, the number of neurites emerging from the soma, and the numbers of branches per longest neurite were measured.
3
Quantitative Analysis of Neuronal Morphology
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Images of culture experiments were taken with a confocal laser-scanning microscope (TCS SP8; Leica Microsystems) using HC PL APO CS2 20×/0.75 NA multiple immersion lens (Leica Microsystems) or HC PL APO CS 10×/0.40 NA multiple immersion lens (Leica Microsystems) under constant conditions in terms of laser power, pinhole size, gain, z-steps, and zoom setting throughout the experiments. All quantitative measurements were performed with ImageJ 1.52a software [42] . For the quanti cation of MAP2 and tau staining areas, z-series of optical sections were projected by the brightest point method.
MAP2 and tau staining areas were de ned as area in which staining signal intensites were 2 and 3 times stronger than those of background signals on the same eld, respectively. For analysis of the time course of neurite outgrowth, z-series of optical sections were projected by the brightest point method. Neurite of cortical neurons was identifed by Tuj1 signals, and the length of the longest neurite, the number of neurites emerging from the soma, and the numbers of branches per longest neurite were measured.
MAP2 and tau staining areas were de ned as area in which staining signal intensites were 2 and 3 times stronger than those of background signals on the same eld, respectively. For analysis of the time course of neurite outgrowth, z-series of optical sections were projected by the brightest point method. Neurite of cortical neurons was identifed by Tuj1 signals, and the length of the longest neurite, the number of neurites emerging from the soma, and the numbers of branches per longest neurite were measured.
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