Stereo investigator software

Manufactured by MicroBrightField

Sourced in United States, Germany, Canada, Japan

Stereo Investigator is a software application designed for comprehensive quantitative analysis of microscopic samples. It provides a suite of tools for stereological measurements, tracing, and reconstruction of neural structures.

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Lab products found in correlation

Axiophot photomicroscope, by Zeiss (6 mentions) Sigmafast dab peroxidase substrate, by Merck Group (5 mentions) E800 microscope, by Nikon (5 mentions) Retiga 2000r camera, by MicroBrightField (4 mentions) Abc reagent, by Vector Laboratories (4 mentions) Lsm800 confocal microscope, by Zeiss (4 mentions) Bx51 microscope, by Olympus (3 mentions) Eclipse 80i microscope, by Nikon (3 mentions) Hv c20 camera, by MicroBrightField (3 mentions) Eclipse e600 microscope, by Nikon (3 mentions) Bx53 microscope, by Olympus (2 mentions) Bx61 dsu microscope, by Olympus (2 mentions) Eclipse 90i microscope, by Nikon (2 mentions) Microfire digital camera, by Optronics (2 mentions) Zen software, by Zeiss (2 mentions) Mouse anti th, by Santa Cruz Biotechnology (2 mentions) Leica dm2000 microscope, by Leica camera (2 mentions) Axio imager m2, by Zeiss (2 mentions) Hv c20, by Hitachi (2 mentions) Zen 2.3 software system, by Zeiss (2 mentions)

175 protocols using stereo investigator software

Standardized Morphometric Analysis Pipeline

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All morphometric measures were performed using Stereo Investigator software (version 9.14.5 32-bit, MicroBrightField, Inc., Williston, VT, USA) coupled to a Nikon Optiphot-2 microscope, as described previously (Kelley, et al., 2013 ). To control for inter-individual variability, a single investigator preformed all analyses. To ensure low intra-individual variability, five mice chosen at random were measured non-consecutively three times during each triad (beginning, middle, end) of experiment. No significant difference was found across these time points. All analyses were conducted blinded to genotype and treatment.
Photomicrographs were taken on a Nikon Optiphot-2 microscope (Tokyo, Japan) connected to Stereo Investigator software (MicroBrightField, Inc.) (Figs. 1, 2, 3, 6). Background correction was used at the time of image capture to establish evenness of illumination across the field, and scale bars were added within the Stereo Investigator software. Panels were compiled in PowerPoint (version 14.0.6129.5000, 32-bit, Microsoft, Redmond, WA, USA), and in Figs. 1, 2, and 3 each micrograph was equally corrected for brightness and contrast. No retouching or further manipulations were performed.

Kelley C.M., Powers B.E., Velazquez R., Ash J.A., Ginsberg S.D., Strupp B.J, & Mufson E.J. (2014). Maternal choline supplementation differentially alters the basal forebrain cholinergic system of young-adult Ts65Dn and disomic mice. The Journal of comparative neurology, 522(6), 1390-1410.

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Unbiased Stereological Analysis of Striatal DARPP32+ Cells in Huntington's Disease Rat Model

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Female and male hom, het and control (wt) F344tgHD rats were used at 8 months of age (n = 3, respectively). Brains were removed and processed as described above. Every sixths section (40 μm) including the striatum was stained with anti-DARPP32 antibody (Abcam Cat# ab40801, RRID:AB_731843) for 72 h at 4°C, with two interstitial 8 h periods at room temperature. Anatomical boundaries of the striatum were adapted from Kántor et al. (2006) (link). Z-stacks (1 mm steps) were taken with an Olympus BX51-DSU microscope and an UPLSAPO objective 20x (numerical aperture: 0.75). Unbiased analysis of the number of DARPP32⁺ cells was conducted by an experimenter, blinded to the animals’ genotype and sex with the optical fractionator method/Stereo Investigator^® Software (MicroBrightField, Williston, VT, USA). The height of the counting sites was set to 10 μm with a 3.5 μm guard zone above and below. Grid size was determined as 500 μm × 500 μm with a systematic random sampling and frame size as either 150 μm × 150 μm or 100 μm × 100 μm, depending on the cell density. On average, 311 counting frames were counted manually per animal. Striatal volume was evaluated using the Cavalieri principle/Stereo Investigator^® Software (MicroBrightField, Williston, VT, USA) with 100 μm grid spacing and a randomized grid rotation.

Ratz-Wirsching V., Habermeyer J., Moceri S., Harrer J., Schmitz C, & von Hörsten S. (2024). Gene-dosage- and sex-dependent differences in the prodromal-Like phase of the F344tgHD rat model for Huntington disease. Frontiers in Neuroscience, 18, 1354977.

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Stereological Quantification of Neuronal Populations

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Stereology was performed with the Stereo Investigator software package (version 11.07; MicroBrightField Biosciences, Williston, VT). The investigator was blinded to experimental groups by using coded object slides. Nine SN and four STN sections in average separated by 240μm (one of six series) were used for counting. TH⁺ or NeuN⁺ neurons in both SN pars compacta and reticulata and Nissl⁺ STN neurons were included within each selected region. Counting parameters were: TH, Nissl: grid size 130 × 130μm, counting frame 60 × 60μm, and 1.5‐μm guard zone; NeuN: grid size 200 × 200μm, counting frame 60 × 60μm, and 1.5‐μm guard zone. Actual mounted thickness was determined by randomly selecting sections and determining thickness at every counting site. Sections were viewed under a 100×/1.25 numerical aperture objective (Olympus, Tokyo, Japan) on a BX53 microscope. Gundersen coefficients of error for m = 1 were all less than or equal to 0.09 for each section counted.

Musacchio T., Rebenstorff M., Fluri F., Brotchie J.M., Volkmann J., Koprich J.B, & Ip C.W. (2017). Subthalamic nucleus deep brain stimulation is neuroprotective in the A53T α‐synuclein Parkinson's disease rat model. Annals of Neurology, 81(6), 825-836.

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Quantitative Analysis of Midbrain Neurodegenerative Markers

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Immunostaining method was described in a previous publication [52 (link)]. Detail information about the reagents in this part was shown in Table S1. Primary antibodies including anti-TH, anti-p-α-syn, anti-NF, and anti-GFAP were used either for midbrain or sciatic nerve. Then, secondary antibodies of Alexa Fluor 488 and 594 were incubated. Olympus FV1000 confocal laser scanning microscope was applied to acquire images.
For immunohistochemistry, brain slices were incubated with primary antibody of anti-TH. Number of TH⁺ neurons in SNpc of midbrain was assessed using optical fractionator (Stereo Investigator software, Microbrightfield Bioscience, Williston, VT, USA). All stereological analyses were performed under × 200 magnification of Olympus BX52 microscope (Olympus America Inc., Melville, NY, USA).

Sun L., Jiang W.W., Wang Y., Yuan Y.S., Rong Z., Wu J., Fan Y., Lu M, & Zhang K.Z. (2021). Phosphorylated α-synuclein aggregated in Schwann cells exacerbates peripheral neuroinflammation and nerve dysfunction in Parkinson’s disease through TLR2/NF-κB pathway. Cell Death Discovery, 7, 289.

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Stereological Cell Counting in Frontal and Parietal Cortex

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Stereological cell count was done by a blinded observer to the animals' identity on regularly spaced (1/10) sections (average post-processing thickness was 20 μm) under a 40 × objective with the optical fractionator method using the Stereo-Investigator software (MicroBrightField, Inc., Williston, VT, USA) in the frontal and parietal cortex of one hemisphere. The frontal cortex was analyzed between 1.94 and 0.86 mm anterior and 0 to 2.0 mm lateral from bregma, and the parietal cortex between 1.1 and 2.3 mm posterior and 1.0 to 3.0 mm lateral from bregma. Coordinates were based on Paxinos and Franklin (2012) .

Salama M, & Mohamed W.M. (2016). Tau protein as a biomarker for asphyxia: A possible forensic tool?. Applied & Translational Genomics, 9, 20-22.

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Quantitative Spinal Motor Neuron Analysis

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A separate set of cervical and lumbar spinal cord sections from randomly selected mice from each group (n = 5/group) were stained with 0.1% cresyl violet using a standard protocol for examination of motor neuron condition for the Nissl substance. Motor neuron numbers in the ventral horn of the cervical and lumbar spinal cords were determined by the optical fractionator method of unbiased stereological cell counting techniques using a Nikon Eclipse 600 microscope and quantified by using Stereo Investigator® software (MicroBrightField). The virtual grid (150 × 150 µm) and counting frame (75 × 75 µm) were optimized to count at least 200 cells per animal with error coefficients <0.07. Outlines of the anatomical structures were done using 10X/0.45 objective, and cell quantification was conducted using 40X/1.40 objective. The motor neuron numbers (20–25 μm diameter) were counted in discrete levels of the cervical (C1–C3, C4–C6, and C7–C8) and lumbar spinal (L1–L2, L3–L4, and L5–L6) cords (n = 7 sections/level/spinal cord segment/group separated by approximately 120 μm) and presented as averages per ventral horn for both spinal cord sides. Motor neuron morphologies were also analyzed in the cervical and lumbar spinal cords.

Garbuzova-Davis S., Kurien C., Thomson A., Falco D., Ahmad S., Staffetti J., Steiner G., Abraham S., James G., Mahendrasah A., Sanberg P.R, & Borlongan C.V. (2017). Endothelial and Astrocytic Support by Human Bone Marrow Stem Cell Grafts into Symptomatic ALS Mice towards Blood-Spinal Cord Barrier Repair. Scientific Reports, 7, 884.

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Quantitative Analysis of Cortical Neuron Markers

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Quantitation of NeuN, encephalopsin, Emx-1 and annexin V immunolabeled cortical neurons were obtained using a computer interfaced with an Olympus BX61-DSU microscope with a motorized stage, running StereoInvestigator software (version 10, MicrobrightField), as previously described (40 (link)). In brief, 30-μm serial paraffin sections were placed individually on slides and every third slide was immunolabeled as described above. Sections (n = 5) were analyzed per animal and 3 animals were used per time-point. Regions of interest in layers 1–4 and 5 were traced at low magnification (4× magnification, NA 0.16), within the motor strip including the primary motor cortex, secondary motor cortex, and primary somatosensory hind limb region (Fig. 1A). Counting was performed using random counting grids (50 × 50 μm) and a 40× objective (NA 1.3). For layers 1–4, counting sites ranged between 23–30 and for layer 5, 14–17 sites were counted. The optical dissector and guard zone were set at 20 and 2 μm, respectively. Cell densities were calculated by dividing the averaged total immunolabeled cell number by the product of counting site volume × mean number of total sites and correcting the values as cells per cubic mm. The data obtained from this analysis were tested for significance by the Wilcoxon-Mann Whitney test.

Burns T., Miers L., Xu J., Man A., Moreno M., Pleasure D, & Bannerman P. (2014). Neuronopathy in the Motor Neocortex in a Chronic Model of Multiple Sclerosis. Journal of neuropathology and experimental neurology, 73(4), 335-344.

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Quantifying APP-Positive Particles in Subcortical White Matter

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Stereological measurements were made by using a Nikon Eclipse 90i microscope (Nikon, Tokyo, Japan) attached to a Qimage Retiga-2000R camera, which was connected to a workstation with Stereo Investigator software (Version 10; MicroBrightField, Williston, VT, USA). Every 12th section was examined, placing the analyzed sections 600 μm apart. Using a 2× objective, we traced the entire area of subcortical white matter for outlining the region of interest to be used in the stereological analysis. Then we counted the APP-positive particles under a 40× objective. We identified particles as being a brown dot that first came into focus within the optical dissector counting frame. The counting frame was 150 × 150 μm with a grid size of 3,000 × 3,000 μm. The dissector height was set at 10 μm with an upper and lower guard zone of 1 μm. This analysis used 11–14 coronal sections and 98–176 sampling fields to ensure that at least 100 particles were counted per brain (range 118–1086) to obtain the estimate of the total number of APP-positive particles within the entire subcortical white matter area.

Wang J., Shi Y., Cao S., Liu X., Martin L.J., Simoni J., Soltys B.J., Hsia C.J, & Koehler R.C. (2023). Polynitroxylated PEGylated hemoglobin protects pig brain neocortical gray and white matter after traumatic brain injury and hemorrhagic shock. Frontiers in Medical Technology, 5, 1074643.

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Stereological Analysis of Cell Populations

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The optical fractionator and nucleator probes were used on an Olympus microscope with StereoInvestigator software (MicroBrightField) to defined a systematic-random sampling sequence of counting frames and grids in 100 μm sections within each area analyzed. A section sampling interval of ten was examined. The neuroanatomical regions including layers II–III and V–VI were defined at low magnification with a 2× objective. The optical fractionator probe was run within the selected neuroanatomical region with a 100× oil objective. The population estimate was used to determine the section sampling fraction (0.09), the area sampling fraction was (8.2), and the height sampling fraction (0.66) necessary to reach a coefficient of error equal or less than 0.008 [14 (link)]. To prevent sampling bias, post-processing tissue thickness was measured at each sampling site. Cells were counted if the cellular nucleolus was into focus within the dissector counting frame. The nucleator probe was implemented during the optical fractionator allowing for simultaneous cell number and cell volume estimates. The nucleolus was selected as the midpoint of the cell and the distance from the midpoint to the soma margin was used to calculate soma volume. For stereological parameters go to supplemental table 2.

Kim E., Camacho J., Combs Z., Ariza J., Lechpammer M., Noctor S, & Martínez-Cerdeño V. (2015). Preliminary findings suggest the number and volume of supragranular and infragranular pyramidal neurons are similar in the anterior superior temporal area of control subjects and subjects with autism. Neuroscience letters, 0, 98-103.

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Stereological Quantification of PV and SOM Cells in Nlgn3 Mice

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Unbiased stereological estimation of total PV and SOM cells in the somatosensory cortex of WT and Nlgn3^R451C mice was acquired using the optical fractionator approach and the Stereo Investigator software (MicroBrightField, Inc.) equipped with an Olympus BX51 microscope. Boundaries of the entire somatosensory cortex were determined using anatomical landmarks, the ALLEN Mouse Brain Reference Atlas Version 2 (2011), and Paxinos and Franklin Mouse Brain Atlas (Second Edition) and traced at low magnification (2x, NA 0.05). A sampling grid of 700 μm x 700 μm, counting frame of 100 μm x 100 μm, and dissector height of 10 μm (with 1–3 μm guard zones) were used to estimate the number of positively stained cells in a 1:10 series for each animal at high magnification (40x, NA 0.75). The Gundersen coefficient of error (m = 0) was below 0.1 for all animals (n = 10 for both WT and Nlgn3^R451C groups) (Gundersen and Jensen, 1987). Data are shown as % of the WT average cell number for either PV neurons or SOM neurons in Nlgn3^R451C mice (mean ± SEM). Representative images were taken using the 40x objective on the same Olympus BX51 microscope used for stereology.

Speed H.E., Masiulis I., Gibson J.R, & Powell C.M. (2015). Increased Cortical Inhibition in Autism-Linked Neuroligin-3R451C Mice Is Due in Part to Loss of Endocannabinoid Signaling. PLoS ONE, 10(10), e0140638.

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