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.
Cast stereology software
Manufactured by Olympus
Sourced in Japan
CAST stereology software is a versatile tool designed for image analysis and quantitative assessment of microscopic structures. It provides a robust platform for 3D reconstruction and measurement of various biological and material samples.
Automatically generated - may contain errors
Lab products found in correlation
Bx51 microscope, by Olympus (4 mentions)
Dp70 digital ccd camera, by Olympus (3 mentions)
Bx52 epifluorescent microscope, by Olympus (3 mentions)
Nanozoomer xr digital slide scanner, by Hamamatsu Photonics (2 mentions)
Dp70 digital camera system, by Olympus (2 mentions)
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Iba 1, by Abcam (2 mentions)
Glial fibrillary acidic protein (gfap), by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (2 mentions)
Bx51 epifluorescence microscope, by Olympus (1 mentions)
Lsm510 meta confocal microscope, by Olympus (1 mentions)
8 protocols using cast stereology software
1
Quantitative Analysis of Amyloid Pathology
2
Stereology-based Quantification of Viral Transduction
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Stereology-based studies were performed as previously described,92 (link), 93 (link) using a motorized stage of an Olympus BX51 epifluorescence microscope equipped with a DP70 digital CCD camera, an X-Cite fluorescent lamp, and the associated CAST stereology software version 2.3.1.5 (Olympus, Tokyo, Japan). The cortex was initially outlined under the 4× objective. Random sampling of the selected area was defined using the optical dissector probe of the CAST software. To evaluate the percentage of Anc80L65, AAV9, and scAAV9 transduced astrocytes or neurons, we performed the stereology-based counts under the 20× objective, with a meander sampling of 10% for the surface of cortex for Anc80L65 and scAAV9, and 30% for AAV9 (the EGFP-positive cells being seldom in this case). For each counting frame, the total number of astrocytes (GS-positive cells) or neurons (NeuN-positive cells) was evaluated, and among each of those populations, the percentages of EGFP-positive cells. Only glial and neuronal cells with a DAPI-positive nucleus within the counting frame were considered. Counts were performed blindly.
3
Quantifying Dopaminergic Neurons via Stereology
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For dopaminergic neuron counts, a complete set of serial SN sections at 30 μm from each animal was stained for TH and counterstained for Nissl substance (Chen et al., 2013 (link), Chen et al., 2017 (link)). Sections were coded and the number of TH-positive cells was counted by unbiased stereology based on the optical fractionator principle using Olympus BX51 microscope and Olympus CAST stereology software, as previously described (Dimant et al., 2013 (link); West et al., 1991 (link)).
4
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.
5
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.
6
Stereological Quantification of Dopaminergic Neuron Loss
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Fluorescence images were obtained on either a Zeiss LSM510 META confocal microscope with X20 magnification for fluorescence imaging or an Olympus BX51 microscope for DAB imaging. At least 8 coronal sections per animal throughout the SNpc were analyzed to determine tyrosine hydroxylase cell loss by conducting unbiased stereology cell counting using the Olympus CAST stereology software. An average of 300 cells in the SNpc were analyzed for venusYFP fluorescence. For NeuN lesion analysis, coronal sections were co-stained with anti TH and anti NeuN antibodies. The SNpc was outlined using Fiji (NIH) according to TH staining and was saved as a region of interest. The amount of NeuN positive neurons only within that region was calculated using Fiji's particle analysis feature.
7
Immunocytochemical Assay Protocol for Cellular Analysis
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Cells were seeded in 24-well plates. After treatment, the medium was removed and the plates were washed with PBS three times, the slides were xed with 4% paraformaldehyde for 15 min and washed with PBS three times, and permeabilized with 0.5% Triton X-100 at room temperature for 20 min. Plates were washed with PBS. 5% goat serum was added and incubated at room temperature for 30 min. Primary antibodies were added and incubated at 4°C overnight. Primary antibodies used were microtubule-associated protein 2 (MAP2) (1:500, Thermo Fisher Scienti c), Iba1 (1:500, Abcam, USA), Ki-67 (1:100, Thermo Fisher Scienti c), and glial brillary acidic protein (GFAP) (1:1000, Sigma-Aldrich,) Plates were washed with PBS and incubated with secondary antibody: goat anti-rabbit IgG-Alexa Fluor 488 (1:500, Molecular Probes, USA) or donkey anti-mouse IgG-Alexa Fluor 546 (1:500, Thermo Fisher Scienti c) for 45 min in the dark at room temperature, washed with PBS, and stained with DAPI (Vector Laboratories, USA). For analysis of uorescence intensity, images were captured under an Olympus BX51 microscope (Olympus Optical Co., Tokyo, Japan) with Olympus CAST stereology software and a DP 70 digital camera system using the same camera gain, exposure time and pixel setting for all samples. Integrated optical density (IOD) of staining in the images was analyzed by Image J.
8
Immunocytochemical Assay Protocol for Cellular Analysis
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Cells were seeded in 24-well plates. After treatment, the medium was removed and the plates were washed with PBS three times, the slides were xed with 4% paraformaldehyde for 15 min and washed with PBS three times, and permeabilized with 0.5% Triton X-100 at room temperature for 20 min. Plates were washed with PBS. 5% goat serum was added and incubated at room temperature for 30 min. Primary antibodies were added and incubated at 4°C overnight. Primary antibodies used were microtubule-associated protein 2 (MAP2) (1:500, Thermo Fisher Scienti c), Iba1 (1:500, Abcam, USA), Ki-67 (1:100, Thermo Fisher Scienti c), and glial brillary acidic protein (GFAP) (1:1000, Sigma-Aldrich,) Plates were washed with PBS and incubated with secondary antibody: goat anti-rabbit IgG-Alexa Fluor 488 (1:500, Molecular Probes, USA) or donkey anti-mouse IgG-Alexa Fluor 546 (1:500, Thermo Fisher Scienti c) for 45 min in the dark at room temperature, washed with PBS, and stained with DAPI (Vector Laboratories, USA). For analysis of uorescence intensity, images were captured under an Olympus BX51 microscope (Olympus Optical Co., Tokyo, Japan) with Olympus CAST stereology software and a DP 70 digital camera system using the same camera gain, exposure time and pixel setting for all samples. Integrated optical density (IOD) of staining in the images was analyzed by Image J.
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