Proliferating radial glial cells (Ki67/GFAP positive) within the ipsilateral SVZ and contralateral SVZ were quantified using the cell counter plugin for National Institute of Health ImageJ software (USA). Qualitative images of immature astrocytes (Nestin/GFAP positive) observed extending from the SVZ toward penumbral regions was obtained. Images were obtained using a laser scanning inverted confocal imaging system (Nikon Instruments Inc., Melville, NY, USA) under x40 magnification (a higher magnification was required to achieve greater image resolution for cell counts). For quantification, images of the SVZ were presented as collapsed reconstructions of 10 optical sections captured every 0.5 µm on the z-axis, at a scan speed of 0.5 frame/sec, a resolution of 1024×1024 pixels with the line acquisition mode. Due to a small counting frame within the SVZ cell numbers were quantified using standard stereological techniques and ImageJ. The total number of proliferating radial glial cells detected in the ipsilateral SVZ was then compared to the corresponding contralateral SVZ and expressed as 100% control.
Laser scanning inverted confocal imaging system
Manufactured by Nikon
Sourced in United States
The Laser Scanning Inverted Confocal Imaging System is a highly specialized laboratory equipment designed for advanced microscopy applications. It utilizes a laser-based scanning mechanism and an inverted microscope configuration to capture high-resolution, three-dimensional images of biological samples.
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2 protocols using laser scanning inverted confocal imaging system
1
Quantifying Proliferating Radial Glial Cells in SVZ
2
Quantifying Immature Neurons in SVZ
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Migrating immature neurons or neuroblasts (DCX positive) within the ipsilateral SVZ and contralateral SVZ were quantified using the cell counter plugin for National Institute of Health ImageJ software (USA). DCX positive cells within the ipsilateral SVZ and contralateral SVZ were quantified using the cell counter plugin for National Institute of Health ImageJ software (USA). Images were obtained using an Olympus microscope (Albertslund, Denmark) under x20 magnification (the same magnification utilised with CAST counts). Qualitative images of proliferating immature neurons (Ki67/DCX positive) observed within the SVZ were obtained using a laser scanning inverted confocal imaging system (Nikon Instruments Inc., Melville, NY, USA) under x40 magnification (a higher magnification was required to achieve greater image resolution for cell counts). Images were presented as collapsed reconstructions of 17 optical sections captured every 0.4 µm on the z-axis, at a scan speed of 0.5 frame/sec, a resolution of 1024×1024 pixels with the line acquisition mode. Due to a small counting frame within the SVZ cell numbers were quantified using standard stereological techniques and ImageJ. The total number of immature neurons in the ipsilateral SVZ was then compared to the corresponding contralateral SVZ and expressed as 100% control.
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