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Bx50 stereological microscope

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The BX50 stereological microscope is a versatile laboratory instrument designed for microscopic observation and analysis. It features high-quality optics, including a binocular eyepiece, and provides a clear, magnified view of samples. The BX50 is capable of performing basic microscopic tasks, but a detailed product description cannot be provided while maintaining an unbiased and factual approach.

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Lab products found in correlation

Bx50 microscope, by Olympus (2 mentions) Aperio at2 scanner, by Leica (1 mentions) Iba1 antibody, by Fujifilm (1 mentions) Th antibody, by Merck Group (1 mentions) Vectastain abc reagent, by Vector Laboratories (1 mentions) At2 scanner, by Leica (1 mentions)

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BX50 microscope

6 protocols using bx50 stereological microscope

1

Quantifying Neuronal Subpopulations in the Brain

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Free-floating 35μm coronal sections containing the LC, SNpc, VTA, hippocampus (Hip), motor cortex (MCtx) and caudate putamen (CPu) were cut on a horizontal sliding microtome. Noradrenergic neurons of the LC and dopaminergic neurons of the VTA and SNpc were identified through TH+ immunohistochemistry, whereas the pan-neuronal marker NeuN was used to identify all neurons in the Hip, MCtx and CPu. Stereological counts of TH+ LC, VTA and SNpc neurons were estimated using an optical fractionator method on an Olympus BX50 stereological microscope within user-defined boundaries [20 (link)]. Automated counting was performed on high-density clusters of NeuN+ neurons in the Hip, MCtx and CPu were performed using an automated counting feature on ImageJ [25 (link)]. All immunohistochemistry images were captured by a Leica Aperio AT2 Scanner.
Song S., Jiang L., Oyarzabal E.A., Wilson B., Li Z., Shih Y.Y., Wang Q, & Hong J.S. (2018). Loss of brain norepinephrine elicits neuroinflammation-mediated oxidative injury and selective caudo-rostral neurodegeneration. Molecular neurobiology, 56(4), 2653-2669.
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2

Immunohistochemical Analysis of Dopaminergic and Microglial Markers in LPS-Treated Mice

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For immunohistochemistry, mice (n = 5-6 each group) treated with LPS or saline were perfused transcardially with 4% paraformaldehyde and coronal SN and LC sections were used for immune-staining as described previously (Wang et al., 2015 (link)). We used the following primary antibodies for immunohistochemistry: tyrosine hydroxylase (TH; 1:2,000; EMD Millipore, Temecula, CA) and ionized calcium binding adaptor molecule-1 (Iba-1; 1:5000; Wako Chemicals, Richmond, VA). Immuno-staining was visualized by using 3,3′-diaminobenzidine (DAB) and urea-hydrogen peroxide tablets or nickel-enhanced DAB or Alexa Fluor 488 (green) and 555 (red) dye. Stereological counts of TH+ NE-LC neurons were estimated using an optical fractionator method on an Olympus BX50 stereological microscope within userdefined boundaries (Song et al., 2019a (link)). The quantification of immunohistological staining in different brain regions was performed by Image J software as described previously (Song et al., 2019a (link); Song et al., 2019b (link)). Briefly, the image was first converted into the grayscale picture, and the background was adjusted before the quantifying area was selected for the measurement of the total pixels. The relative density of the staining was compared based on the density of the total pixels of a certain brain region (total pixels/area).
Wang Q., Oyarzabal E.A., Song S., Wilson B., Santos J.H, & Hong J.S. (2020). Locus coeruleus neurons are most sensitive to chronic neuroinflammation-induced neurodegeneration. Brain, behavior, and immunity, 87, 359-368.
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3

Stereological Analysis of Dopaminergic Neurons

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Free-floating 35 μm coronal sections containing the SNpc were cut on a horizontal sliding microtome. A total of 8 sections were sampled at 105 µm intervals for each brain region. The free-floating sections were immune-blocked with 4% goat serum in 0.25% Triton/PBS for 2 h and then incubated with Iba-1 antibody (1:2000, Wako Chemicals, Richmond, VA) overnight at 4 °C. On the second day, the sections were washed by 1% BSA in 0.25% Triton/PBS before the incubation with anti-tyrosine hydroxylase (TH) antibody (1:2000, EMD Millipore, Temecula, CA) overnight at 4 °C. The double-label immunofluorescence pictures were taken under the confocal microscope by using Alexa-488 (green) and Alexa-594 (red) conjugated secondary antibodies (1:1000) to visualize the TH immune reactive (THir) or Iba-1 positive cells. Stereological counts of TH+ SNpc neurons were estimated using an optical fractionator method on an Olympus BX50 stereological microscope within user-defined boundaries. Samples were countered in a double-blind manner. Data were expressed as percentage to saline-injected controls [26 (link), 39 (link)].
Fan X.M., Luo Y., Cao Y.M., Xiong T.W., Song S., Liu J, & Fan Q.Y. (2020). Chronic Manganese Administration with Longer Intervals Between Injections Produced Neurotoxicity and Hepatotoxicity in Rats. Neurochemical Research, 45(8), 1941-1952.
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4

Stereological Counting of Dopaminergic Neurons

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Dopaminergic neurons of the SNpc were identified through TH immunohistochemistry. The number of TH-immunoreacted (TH-ir) neurons in SNpc between saline and LPS-injected groups was counted with both optical fractionator and manual counting ways. For stereological counts, a total of eight sections containing the SNpc were sampled at 105-μm intervals. We used an optical fractionator method on an Olympus BX50 stereological microscope to estimate TH-ir neurons within user-defined boundaries [21 (link)–23 (link)]. Systematic random sampling of sites with an unbiased counting frame (100 μm × 100 μm) was conducted within defined boundaries of the SN. The guard zone was set at 2 μm and the dissector height was 11 μm. The counts were performed with an Olympus BX50 microscope with a 60 × 1.4NA oil immersion objective. The coefficient of error values was less than 0.1. For manual count, three individuals performed counting in a double-blind manner [46 (link)].
Zhao Z., Wang Y., Zhou R., Li Y., Gao Y., Tu D., Wilson B., Song S., Feng J., Hong J.S, & Yakel J.L. (2020). A novel role of NLRP3-generated IL-1β in the acute-chronic transition of peripheral lipopolysaccharide-elicited neuroinflammation: implications for sepsis-associated neurodegeneration. Journal of Neuroinflammation, 17, 64.
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5

Quantification of Dopaminergic Neurons in Mouse Brain

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Immunostaining was performed as described previously24 (link). Mouse brains were cut into 35 µm sections on a horizontal sliding microtome. The free-floating sections were immune-blocked with 4% goat serum in 0.25% triton/PBS for 1 h. Dopaminergic neurons were stained with anti-tyrosine hydroxylase (TH, 1:4,000, overnight at 4 °C). Sections were incubated with biotinylated secondary antibody for 1 h followed by incubation with Vectastain ABC reagents (Vector Labs, Burlingame, CA) for 40 min and then color was developed with 3,3-diaminobenzidine. To monitor DA neuro degeneration, two individuals blind to the treatment counted the number of TH-immunoreactive (TH-IR) neurons in the SN pars compacta (SNpc) of six evenly spaced brain sections from a series of 24 sections that covered the entire SN26 (link). Stereological counts of THir SNpc neurons were estimated using an optical fractionator method on an Olympus BX50 stereological microscope within user-defined boundaries24 (link). All immunohistochemistry images were captured by a Leica Aperio AT2 Scanner.
Song S., Liu J., Zhang F, & Hong J.S. (2020). Norepinephrine depleting toxin DSP-4 and LPS alter gut microbiota and induce neurotoxicity in α-synuclein mutant mice. Scientific Reports, 10, 15054.
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6

Stereological Quantification of Dopaminergic Neurons

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Free-floating 35 μm coronal sections containing the SNpc, VTA, hippocampus, motor cortex and caudate/putamen (CPu) were cut on a horizontal sliding microtome. A total of 8 sections were sampled at 105 μm intervals for each brain region. Dopaminergic neurons of the VTA and SNpc were identified through TH+ immunohistochemistry, whereas the pan-neuronal marker NeuN was used to identify all neurons in the hippocampus, motor cortex and CPu. Stereological counts of TH+ VTA and SNpc neurons were estimated using an optical fractionator method on an Olympus BX50 stereological microscope within user-defined boundaries (Song et al., 2018 ; Wang et al., 2014 (link); Wang et al., 2015 (link)). Briefly, systematic random sampling of sites with an unbiased counting frame (100 μm × 100 μm) within defined boundaries of the SN was performed. An 11 μm dissector height was used, and the guard zone was set at 2 μm. The counts were conducted with an Olympus BX50 microscope using a 60× 1.4NA oil immersion objective, and the coefficient of error values were less than 0.1.
Song S., Wang Q., Jiang L., Oyarzabal E., Riddick N.V., Wilson B., Moy S.S., Shih Y.Y, & Hong J.S. (2019). Noradrenergic dysfunction accelerates LPS-elicited inflammation-related ascending sequential neurodegeneration and deficits in non-motor/motor functions. Brain, behavior, and immunity, 81, 374-387.
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