We used StereoInvestigator 10 (Microbrightfield) interfaced with a Nikon Eclipse 80i microscope and an MBF Bioscience 01-MBF-2000R-F-CLR-12 Digital Camera (Color 12 BIT). A total of six counting regions in the vermis and four counting regions in the cerebellar hemisphere were assessed (Figure 1 ). The counting regions represent the anterior, posterior, dorsal and ventral areas of each part of the cerebellum. Four cerebellar sections from the vermis and each cerebellar hemisphere were quantified per animal with a physical distance of 225 µm between them, reducing the likelihood of double counting microglia due to their small cell size. The optical fractionator probe method was used to estimate cell and phagocytic cup densities following stereological parameters previously standardized in our lab (5 ). Iba1+ cells and phagocytic cups were counted at 20X magnification. The overall estimated volume of each counting region was used to normalize counts to obtain an estimation of the average density of objects of interest (e.g., phagocytic microglia, phagocytic cups), which was expressed as number/µm3 (relative density measurement). Experimenters were blinded to the experimental condition of the samples.
Stereo investigator 10
Manufactured by MicroBrightField
Sourced in Japan
Stereo Investigator 10.0 is a software application designed for quantitative analysis of three-dimensional (3D) structures in microscopic samples. It provides a suite of tools for visualizing, navigating, and measuring various morphological features within a 3D environment.
Automatically generated - may contain errors
Lab products found in correlation
Orca flash4.0 lt digital cmos camera, by Hamamatsu Photonics (2 mentions)
Eclipse 80i microscope, by MBF Biosciences (1 mentions)
Dab kit, by Vector Laboratories (1 mentions)
Axio imager a1 microscope, by Zeiss (1 mentions)
Vectastain elite abc kit, by Vector Laboratories (1 mentions)
Axio imager m2 imaging microscope system, by Zeiss (1 mentions)
5 protocols using stereo investigator 10
1
Stereological Quantification of Cerebellar Microglia
2
Quantifying Midbrain Dopaminergic Neurons
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Unbiased stereology was performed to estimate the number of midbrain DAergic neurons62 (link),63 (link). According to the mouse brain in stereotaxic coordinates, a series of 40-μm-thick coronal sections across the midbrain (every fourth section from Bregma −2.54 to −4.24 mm, ten sections per case) were stained with an antibody specific to TH (Pel-Freez, #P40101-150, 1:2500) and subsequently visualized with Vectastain Elite ABC Kit and DAB Kit (Vector Laboratories). Bright-field images were captured by Axio microscope Imager A1 (Zeiss). The number of TH-positive neurons was assessed using the optical fractionator function of Stereo Investigator 10 (MicroBrightField). Four or more mice were used per genotype at each time point. Counters were blinded to the genotypes of the samples. The sampling scheme was designed to have a coefficient of error <10% in order to obtain reliable results.
3
Quantitative Microscopy for Cerebral Cortex
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A Zeiss Axio Imager M2 imaging microscope system (Carl Zeiss, Inc., Jena, Germany) and ORCA-Flash4.0 LT+ Digital CMOS camera (Hamamatsu, Hamamatsu, Japan) connected with computer software Stereo Investigator 10.0 (MicroBrightField, Inc., Williston, VT, USA) was used for image acquisition for the purpose of quantification. Twelve fields within each cerebral cortical sample from each slide were first randomly selected based upon the DAPI channel. The randomly selected RGB images of the cerebral cortex were acquired with cy3 (Red), FITC (Green), and DAPI (Blue) filters, using a 40× objective from the Zeiss Axio Imager M2 imaging microscope system (Figure 2 ) and ORCA-Flash4.0 LT+ Digital CMOS camera (Figure 3 , Figure 4 , Figure 5 , Figure 6 , Figure 7 and Figure 8 ). Each wavelength was acquired separately using the same camera settings for each channel and then pseudo-merged into an RGB image for analysis in Adobe Photoshop. The optical conditions were rigorously maintained the same for all the analysis sessions.
4
Stereological Analysis of SNc Cells
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The total numbers of TH-, GFAP-, and Iba1-positive cells in unilateral SNcs were measured stereologically using an optical fractionator method as previously described [20 (link)–22 (link)]. Every fourth section through the SNc was analysed using Stereo Investigator software (Stereo Investigator 10.0; MicroBrightField, Williston, VT, USA). Immunolabelled cells were counted by the optical fractionator method (× 40 objective; counting frame, 100 × 100 μm; sampling grid, 200 × 200 μm; counting frame thickness, 10 μm). The coefficient of error (Gundersen, m = 1) for cell count estimation was less than 0.15 for each animal.
5
Quantifying Cerebral Vascular Integrity
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An ORCA-Flash4.0 LT+ Digital CMOS camera (Hamamatsu, Hamamatsu, Japan) connected to the computer software Stereo Investigator 10.0 (MicroBrightField, Inc., Williston, VT, USA) was used for image acquisition and subsequent quantification. Six fields from the cerebral cortex on each side of the brain were randomly selected and obtained at 10× magnification to determine the laminin density. Ten fields were collected at 40× magnification to examine vascular pericyte and astrocyte coverage and claudin-5 expression. In addition, two fields from the hippocampus from each side of the brain were randomly selected and obtained at 10× magnification to examine laminin density and seven at 40× magnification to examine vessel coverage and claudin-5 expression. Likewise, four fields from areas of white matter on each side of the brain were randomly selected and obtained at 10× magnification to determine the laminin density and five fields at 40× magnification were collected to examine vessel coverage and claudin-5 expression.
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