Neurite staining was performed using mouse monoclonal [2G10] to neuron specific β III tubulin primary antibody and goat-anti-mouse IgG- H&L (CY3.5) secondary antibody (AbCam, Cambridge, MA). Receptor staining was done with rabbit polyclonal anti-Plexin A4 primary antibody or rabbit polyclonal anti-Eph receptor A4 primary antibody and donkey anti-rabbit (Dylight 488) IgG- H&L secondary antibody (AbCam). Blocking was performed for one hour with 5% BSA in PBS for the primary antibody and 5% goat or donkey serum for the secondary antibody. Primary and secondary staining was carried out in PBS with 0.1% Saponin and 2% BSA for 48 hours, followed by three washes in PBS with 0.1% Saponin. Image acquisition used a Nikon AZ100 stereo zoom microscope (Nikon, Melville, NY) for conventional fluorescence and a Zeiss LSM 510 Meta microscope (Zeiss, Oberkocken, Germany) for confocal fluorescence. Image J (National Institutes of Health, Bethesda, MD) was used for post processing of images.
Az100 stereo zoom microscope
Manufactured by Nikon
The Nikon AZ100 stereo zoom microscope is a high-performance optical instrument designed for a wide range of applications. It features a zoom ratio of 10:1, providing users with the ability to observe specimens at varying magnifications. The microscope is equipped with a binocular viewing head and offers a comfortable working distance, allowing for convenient sample manipulation and examination.
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Lab products found in correlation
2 protocols using az100 stereo zoom microscope
1
Fluorescent Microscopy of Neuron Markers
2
Microscopy Imaging of Neuronal Growth
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Bright field and conventional fluorescent images were acquired with a Nikon AZ100 stereo zoom microscope using 1× and 2× objectives (Nikon, Melville, NY), while confocal images were taken using a Leica TCS SP2 laser scanning microscope and 20× objective (Leica Microsystems, Buffalo Grove, IL). Confocal z-stacks were acquired through the maximum depth of visible neurite growth with thicknesses ranging between 55-65 μm imaged over 20 slices, each 512 × 512. Image processing was performed using ImageJ (National Institutes of Health, Bethesda, MA). For color coding depth in confocal z-stacks, the Z Code Stack function with a Rainbow LUT was applied using the MacBiophotonics Plugin package for ImageJ. Projections of z-stacks were taken as maximum intensity projections. V3D-Viewer software (Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, VA) allowed 3D rendering and visualization of the confocal z-stack images.
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