Fluorescent images were captured using a C2+ laser scanning confocal microscope (Nikon Instruments Inc., Melville, NY). For each animal, images were taken at the SCI epicenter (determined by lesion volume estimates and visually confirmed by GFAP staining) as well as 0.6mm and 1.2mm, rostral and caudal to the epicenter. A montaged maximum-intensity projection image, consisting of 9 adjacent z-stacks, was generated for each tissue section. When necessary, laser powers were adjusted to capture the full fluorescent intensity range of lightly stained sections. To quantify the proportional area of activated microglia/macrophages, threshold-based analysis of IBA-1 immunoreactivity was performed as described previously with modifications (Donnelly et al., 2009 (link)). Captured RGB images were opened in the MetaMorph analysis program (Molecular Devices, Sunnyvale, CA) and the perimeter of each section was digitally outlined referencing the cytoarchitecture revealed by GFAP and IBA-1 staining. Next, the AF546 channel was isolated and a threshold selected which identified positive IBA-1 staining above background. For each tissue section, the density of IBA-1 staining was calculated as the ratio of immunoreactive area/region of interest area (entire cross section).
Metamorph analysis program
Manufactured by Molecular Devices
Sourced in United States
MetaMorph is a comprehensive image analysis software program that enables users to capture, process, analyze, and manage digital microscope images. The software provides a suite of tools for quantifying various features and parameters within microscopic samples.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using metamorph analysis program
1
Quantifying Activated Microglia/Macrophages in SCI
2
Neurite Outgrowth Quantification Protocol
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Neurite growth was assessed for the entire neuronal population. Cultures were stained with β-tubulin III for visualization of neurites. For each experimental condition, images of 50 neurons were recorded for each animal (n = 3) and imported into NeuronJ, an ImageJ plugin (National Institute of Health, USA).
The length of the longest neurite, total neurite outgrowth, and number of branches was also measured; images of 50 neurons were recorded for each animal (n = 3) and imported into the MetaMorph analysis program (Molecular Devices, USA). The number of neurons with neurites was also recorded.
The length of the longest neurite, total neurite outgrowth, and number of branches was also measured; images of 50 neurons were recorded for each animal (n = 3) and imported into the MetaMorph analysis program (Molecular Devices, USA). The number of neurons with neurites was also recorded.
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