Stereoinvestigator system

Manufactured by MicroBrightField

Sourced in United States, Germany

The StereoInvestigator system is a software application designed for high-performance stereological analysis of histological samples. It facilitates the systematic sampling and quantification of 3D structures within tissue sections.

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Lab products found in correlation

Neurolucida software, by MicroBrightField (4 mentions) Vibratome, by Leica (3 mentions) Color digital video camera, by Optronics (2 mentions) E600 microscope, by Nikon (2 mentions) Stereoinvestigator system, by Optronics (2 mentions) Image pro plus 6, by Media Cybernetics (1 mentions) Rabbit anti iba1 antibody, by Fujifilm (1 mentions) Fluorescence microscopy, by Olympus (1 mentions) Mouse anti th antibody, by Merck Group (1 mentions) 80i microscope, by Nikon (1 mentions) Stereo investigator software, by MicroBrightField (1 mentions) Mouse anti tyrosine hydroxylase, by Merck Group (1 mentions) Biotinylated goat anti mouse igg, by Jackson ImmunoResearch (1 mentions) Mouse anti parvalbumin pv, by Merck Group (1 mentions) Avidin biotin complex system, by Vector Laboratories (1 mentions) Mouse anti neun, by Merck Group (1 mentions) Image pro plus, by Media Cybernetics (1 mentions) Fd rapid golgistain kit, by FD NeuroTechnologies (1 mentions) Anti iba1, by Fujifilm (1 mentions) Anti cd68, by Bio-Rad (1 mentions)

37 protocols using stereoinvestigator system

Unbiased Stereological Analysis of Hippocampal PV Interneurons

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Sections processed for immunohistochemistry were used for unbiased estimates of dorsal hippocampus PV interneuron total numbers using the Stereo Investigator System (MicroBrightField Europe e.K.), as described in [36 (link)]. For unbiased estimates of hippocampal PV interneuron total numbers, we applied an optical fractionator stereological design (monolateral count) using the Stereo Investigator System (MicroBrightField Europe e.K.). A stack of MAC 5000 controller modules (Ludl Electronic Products, Ltd) was interfaced with an Olympus BX50 microscope with a motorized stage and a HV-C20 Hitachi digital camera with a Pentium II PC workstation. A 3D optical fractionator counting probe (x, y, z dimension of 50 × 50 × 25 μm) was applied. The hippocampus was outlined using a × 5 objective and cells were marked with a × 40 objective. Data collection was done by a researcher blind to the genotype and gender of each animal. PV interneuron numbers were estimated according to the formula:

N = SQ * \frac{1}{ssf} * \frac{1}{asf} * \frac{1}{tsf}

where SQ represents the number of neurons counted in all optically sampled fields of the hippocampus, ssf is the section sampling fraction, asf is the area sampling fraction, and tsf is the thickness sampling fraction.

Nobili A., Krashia P., Cordella A., La Barbera L., Dell’Acqua M.C., Caruso A., Pignataro A., Marino R., Sciarra F., Biamonte F., Scattoni M.L., Ammassari-Teule M., Cecconi F., Berretta N., Keller F., Mercuri N.B, & D’Amelio M. (2018). Ambra1 Shapes Hippocampal Inhibition/Excitation Balance: Role in Neurodevelopmental Disorders. Molecular Neurobiology, 55(10), 7921-7940.

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Stereological Analysis of Microglia and Dopaminergic Neurons

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Quantification of Iba1⁺ cells in dorsal striatum was carried out according to our previous study [14 (link)] and analyzed with Image-Pro Plus 6.0 (Media Cybernetics, USA).
Total number of TH⁺ neurons in the SNpc was quantified using a Stereo Investigator system (Micro Brightfield, USA), as described previously [14 (link)]. Stereological counting of microglia in the SNpc was carried out on midbrain sections using a rabbit anti-Iba1 antibody (1:1000; Wako, Japan). Mouse anti-TH antibody (1:500; Sigma, USA) was used to identify the SNpc as described by Martin et al. [15 (link)]. Staining was visualized using dye-conjugated secondary antibodies as in the method of immunofluorescence staining. Iba1⁺ cells were counted using a fluorescence microscopy (Olympus, Japan) and the Stereo Investigator system (Micro Brightfield, USA). The experiment was performed in a double-blind fashion.

Wang Z., Dong H., Wang J., Huang Y., Zhang X., Tang Y., Li Q., Liu Z., Ma Y., Tong J., Huang L., Fei J., Yu M., Wang J, & Huang F. (2020). Pro-survival and anti-inflammatory roles of NF-κB c-Rel in the Parkinson's disease models. Redox Biology, 30, 101427.

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Hippocampal and Amygdalar Volume Measurements

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Volume measurements were performed with a 4X Plan Fluor objective (N.A. 0.13) on a Nikon 80i microscope linked to the PC-based StereoInvestigator system (MicroBrightField, Williston, VT), according to the Cavalieri principle (Gundersen & Jensen, 1987 (link)). About 30 sections per animal (480 μm apart) were used for the measurements of the dentate gyrus; about 15 sections per animal (960 μm apart) were used for CA3, CA2, CA1, and subiculum. About 15 sections per animal (480 μm apart) were used for the amygdala nuclei. We chose to leave out the most rostral and caudal sections of certain fields in order to avoid a biased sampling resulting from the tangential cut of these extreme regions in coronal sections. The first section of the hippocampus was selected randomly within the first two sections through the dentate gyrus, and the last section was selected based on the presence of a clearly identifiable granule cell layer. The first section of the amygdala was selected based on the clear presence of the lateral, basal and paralaminar nuclei, and the last section based on the presence of the medial nucleus.

Villard J., Bennett J.L., Bliss-Moreau E., Capitanio J.P., Fox N.A., Amaral D.G, & Lavenex P. (2021). Structural differences in the hippocampus and amygdala of behaviorally inhibited macaque monkeys. Hippocampus, 31(8), 858-868.

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Stereological Quantification of PV+ and NPY+ Interneurons in Hippocampal Subfields

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Numbers of PV+ or NPY+ interneurons were stereologically counted for the DG, CA1 and CA3 subfields of the hippocampus. All counts utilized every 15th or 20th section through the entire septo-temporal axis of the hippocampus and the optical fractionator method available in the StereoInvestigator system (Microbrightfield Inc.). The StereoInvestigator system consisted of a color digital video camera (Optronics Inc.) interfaced with a Nikon E600 microscope. A detailed protocol employed for cell counting using the optical fractionator has been previous described (Megahed et al., 2014 (link)).

Hussain T., Kil H., Hattiangady B., Lee J., Kodali M., Shuai B., Attaluri S., Takata Y., Shen J., Abba M.C., Shetty A.K, & Aldaz C.M. (2018). Wwox deletion leads to reduced GABA-ergic inhibitory interneuron numbers and activation of microglia and astrocytes in mouse hippocampus. Neurobiology of disease, 121, 163-176.

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Stereological Quantification of Parvalbumin-Positive Cells

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Quantitative observations were limited to the dorsal striatum of the left hemisphere. Using the Stereo Investigator System (MicroBrightField Europe e.K., Magdeburg, Germany) composed of a Zeiss Axioimager.M2 microscope and MicroBrightField’s Stereo Investigator software package, an optical fractionator, stereological design was applied to obtain unbiased estimates of total PV+ cells. Sampling grids and magnifications were adjusted to obtain a relatively constant number of cells sampled and a coefficient of error (CE Gunderson) of ≤0.1. A tri-dimensional optical dissector counting probe (x, y, z dimension of 200 × 200 × 25 μm, respectively) was applied. Counts were performed using a ×20 objective.
Total number was estimated according to the following formula:

N = \sum Q \times 1 / s s f \times 1 / a s f \times 1 / t s f .

where ΣQ represents the total number of neurons counted in all optically sampled fields of the dorsal striatum, ssf is the section sampling fraction, asf is the area sampling fraction, and tsf is the thickness sampling fraction.

Gentile A., Musella A., Bullitta S., Fresegna D., De Vito F., Fantozzi R., Piras E., Gargano F., Borsellino G., Battistini L., Schubart A., Mandolesi G, & Centonze D. (2016). Siponimod (BAF312) prevents synaptic neurodegeneration in experimental multiple sclerosis. Journal of Neuroinflammation, 13(1), 207.

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Neuronal Immunostaining of Brain Sections

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Coronal sections of the brain were cut at a thickness of 30 µm using a vibrating microtome (vibratome 1000, the Vibratome Company, St. Louis, MO, USA). Primary antibodies, including mouse anti-tyrosine hydroxylase (TH; 1:2000; Sigma-Aldrich) or mouse anti-Parvalbumin (PV) (1:2000; Sigma-Aldrich), were used. Biotinylated goat anti-mouse IgG (1:500 the Jackson ImmunoResearch Laboratories, West Grove, PA, USA) was used as the secondary antibody. Immunoreactivity was amplified using an avidin-biotin complex system (1:100, Vector Labs, Burlingame, CA, USA). TH is the key enzyme for dopamine synthesis, and TH immuno-histochemistry was used to label dopaminergic neurons. PV immunostaining was used to label PV-positive GABAergic interneurons. The densities of immunopositive cells were estimated using StereoInvestigator system (MicroBrightField Bioscience, Williston, VT, USA) [35 (link)].

Chang Y.C., Li W.Y., Lee L.J, & Lee L.J. (2020). Interplay of Prenatal and Postnatal Risk Factors in the Behavioral and Histological Features of a “Two-Hit” Non-Genetic Mouse Model of Schizophrenia. International Journal of Molecular Sciences, 21(22), 8518.

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Quantifying Adult Neurogenesis Using Stereology

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All cell counts were accomplished via the optical fractionator method available in the StereoInvestigator system (Microbrightfield) interfaced with a Nikon E600 microscope through a color digital video camera (Optronics Inc., Muskogee, OK). The numbers of newly born BrdU + cells and immature DCX + neurons in the subgranular zone-granule cell layer (SGZ-GCL) were quantified (n = 6/group). The detailed procedure employed for stereological cell counting is available in our previous reports (Rao and Shetty, 2004 (link); Hattiangady et al., 2008 (link); Kodali et al., 2018 (link)). The extent of neuronal differentiation of newly born cells and net neurogenesis was measured through BrdU and neuron-specific nuclear antigen (NeuN) dual immunofluorescence and Z-section analysis in a Nikon confocal microscope (n = 6/group). The primary and secondary antibodies comprised mouse anti-NeuN (1:1000, EMD Millipore, Temecula, CA), rat anti-BrdU (1:250, Serotech), donkey anti-mouse IgG tagged with Alexa Flour 488 (1:200, Invitrogen, Grand Island, NY), and donkey anti-rat IgG tagged with Alexa Flour 594 (1:200, Invitrogen). Percentages of BrdU + cells that expressed NeuN were then quantified by examination of individual BrdU + cells in 1-μm thick optical Z-sections.

Kodali M., Mishra V., Hattiangady B., Attaluri S., Gonzalez J.J., Shuai B, & Shetty A.K. (2021). Moderate, intermittent voluntary exercise in a model of Gulf War Illness improves cognitive and mood function with alleviation of activated microglia and astrocytes, and enhanced neurogenesis in the hippocampus. Brain, behavior, and immunity, 97, 135-149.

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Stereological Analysis of Dopaminergic Neurons

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Densitometric analysis of TH-positive fibers in the striatum was performed as previously described [37 (link)]. An average of 8 sections from the striatum (bregma +1.18 mm to +0.02 mm; anteroposterior axis) were examined using Image-Pro Plus (vision 6.0, Media Cybernetics) on a computer attached to a light microscope (Leica, Germany).
To measure the density of TH-positive cells in the SNpc we performed stereological cell counting as previously described [19 (link), 37 (link)]. Total numbers of TH-positive neurons and Nissl-stained neurons in the SNpc were counted using the optical fractionator method on a Stereo Investigator system (Micro Brightfield, USA) attached to a Leica microscope. A total of 8 sections (one every five 30 μm-thick sections collected −2.80 to −3.80 mm from bregma) were analyzed. Assays were performed in a double-blind fashion by two operators.

Huang D., Li Q., Wang Y., Liu Z., Wang Z., Li H., Wang J., Su J., Ma Y., Yu M., Fei J, & Huang F. (2019). Brain-specific NRSF deficiency aggravates dopaminergic neurodegeneration and impairs neurogenesis in the MPTP mouse model of Parkinson’s disease. Aging (Albany NY), 11(10), 3280-3297.

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Quantifying Substantia Nigra Neurons

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The total numbers of tyrosine hydroxylase-positive (TH⁺) neurons in the SNpc were counted using the optical fractionator method on a Stereo Investigator system (Micro Brightfield, USA) attached to an (Olympus, Japan) as previously described (Liberatore et al., 1999 (link)). Briefly, one out of four 30 μm-thick sections and a total of six sections from bregma –2.80 to –3.65 mm were collected. The SN region was delineated using a 5× objective, and the actual counting was performed under a 40× objective. Stereological counting was performed in a double-blind fashion by two operators.

Bai X., Wang J., Zhang X., Tang Y., He Y., Zhao J., Han L., Fang R., Liu Z., Dong H., Li Q., Ge J., Ma Y., Yu M., Sun R., Wang J., Fei J, & Huang F. (2022). Deficiency of miR-29a/b1 leads to premature aging and dopaminergic neuroprotection in mice. Frontiers in Molecular Neuroscience, 15, 978191.

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Dendritic Spine Density Analysis in Pyramidal Cells

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Dendritic spines were counted in layer II/III pyramidal cells in the OFC using the Golgi-Cox stain. In brief, brain tissues were taken and placed in the impregnation solution (solutions A and B in FD Rapid GolgiStain kit, FD NeuroTechnologies, Ellicott City, MD, USA) for 17 days. After several washes, brain tissues were cut into coronal sections with a vibratome to a thickness of 150 μm. Brain sections were collected and reacted with the mixture of developer and fixer in FD Rapid GolgiStain kit (solutions C and D, 1:1) for 2 minutes. Dendritic spines were examined with a 100× objective oil immersion lens and captured with the Stereoinvestigator system (Microbrightfield, Williston, VT USA). For quantitative analysis of spine density, the spines were counted along dendritic segments chosen from secondary and tertiary dendrites using the Image J software. The experimenter was blind to genotype and selected one to three segments for each cell.

Jiang-Xie L.F., Liao H.M., Chen C.H., Chen Y.T., Ho S.Y., Lu D.H., Lee L.J., Liou H.H., Fu W.M, & Gau S.S. (2014). Autism-associated gene Dlgap2 mutant mice demonstrate exacerbated aggressive behaviors and orbitofrontal cortex deficits. Molecular Autism, 5, 32.

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