For the quantification of amyloid load, Alexa-568-anti-Amyloid immunolabeled and Methoxy-XO4 positive plaques were imaged using a NanoZoomer-XR Digital slide scanner (Hamamatsu Photonics, Shizuoka, Japan) under a 20X objective. The total surface of amyloid was determined using a custom-written script based on the “Analyze particle” function of Fiji (National Institutes of Health: http://fiji.sc/ ), after defining the cortex as region of interest. The total surface occupied by amyloid was then reported to the cortical area of each section considered. Stereology-based study of amyloid-associated microglia was performed on immunolabeled sections using an Olympus BX52 epifluorescent microscope equipped with motorized stage, DP70 digital CCD camera, and CAST stereology software (Olympus, Tokyo, Japan). The cortex was outlined and microglia counts were made using 20X high numerical aperture (1.2) objective. Using a meander sampling of 70% of cortical area, images were captured each time an amyloid deposit was encountered. Those images were then analyzed using Fiji, counting the number of Iba1-positive microglial cells close to a plaque (< 50 μm) and reporting this number to the surface of the plaque considered. All pathology quantification was carried out blinded until the last statistical analyses.
Bx52 epifluorescent microscope
Manufactured by Olympus
Sourced in Japan
The BX52 is an epifluorescent microscope manufactured by Olympus. It is designed to perform fluorescence microscopy, a technique that allows the visualization of specific cellular structures or molecules labeled with fluorescent dyes. The BX52 provides high-quality optical performance and illumination for fluorescence imaging applications.
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3 protocols using bx52 epifluorescent microscope
1
Quantitative Analysis of Amyloid Pathology
2
Quantitative Analysis of Amyloid Pathology
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For the quantification of amyloid load, Alexa-568-anti-Amyloid immunolabeled and Methoxy-XO4 positive plaques were imaged using a NanoZoomer-XR Digital slide scanner (Hamamatsu Photonics, Shizuoka, Japan) under a 20X objective. The total surface of amyloid was determined using a custom-written script based on the “Analyze particle” function of Fiji (National Institutes of Health: http://fiji.sc/ ), after defining the cortex as region of interest. The total surface occupied by amyloid was then reported to the cortical area of each section considered. Stereology-based study of amyloid-associated microglia was performed on immunolabeled sections using an Olympus BX52 epifluorescent microscope equipped with motorized stage, DP70 digital CCD camera, and CAST stereology software (Olympus, Tokyo, Japan). The cortex was outlined and microglia counts were made using 20X high numerical aperture (1.2) objective. Using a meander sampling of 70% of cortical area, images were captured each time an amyloid deposit was encountered. Those images were then analyzed using Fiji, counting the number of Iba1-positive microglial cells close to a plaque (< 50 μm) and reporting this number to the surface of the plaque considered. All pathology quantification was carried out blinded until the last statistical analyses.
3
Stereology-based Quantification of Neurons and Astrocytes
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Stereology-based studies were performed on immunolabeled sections using an Olympus BX52 epifluorescent microscope equipped with motorized stage, DP70 digital CCD camera, and CAST stereology software (Olympus, Tokyo, Japan). The cortex was outlined under low power objective (4×) and astrocytes and neurons counts were made using a 20 × 0.75 numerical aperture objective, with a meander sampling of the selected cortical area. We generally used different probes in order to quantify the overall density of neurons and astrocytes (probe = 5%) and the amount of GFP-positive neurons or astrocytes (probe = 10%). Estimates of the numbers of transduced neurons and astrocytes (by immunolabeling and morphology) were calculated using the fractionator method.
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