The protocol was adapted from the previous investigation [22 (link)]. Briefly, mice were deeply anesthetized and transcardially perfused with PBS followed by 4% paraformaldehyde and 0.05% glutaraldehyde in 0.1 M PB. The spinal cords were removed and postfixed in the same fixative for 4 h at 4 °C. Serial coronal sections at 100 μm thicknesses (VS1000s, Leica, Heidelberger, Germany). The 0.05% Triton X-100 was used for CCR2 immunogold-silver cytochemistry. After rinsing, the sections were postfixed in 2% glutaraldehyde in PBS for 45 min. Signals of CCR2 immunoreactivity were detected by silver enhancement kit in the dark (HQ Silver Kit, Nanoprobes). Sections were postfixed in 0.5% osmium tetroxide in 0.1 M PB for 2 h. They were dehydrated with graded ethanol, replaced with propylene oxide, and finally embedded in Epon 812 between plastic sheets. Flat-embedded sections were examined under the light microscope. The sections containing CCR2 immunoreactivity in spinal dorsal horn were cut with a diamond knife (Diatome, Hatfield, PA). After counterstaining with uranyl acetate, ultrathin sections were examined under the JEM-1230 electron microscope (JEOL LTD, Tokyo, Japan). Postsynaptic and presynaptic membrane, synaptic vesicles were measured using ImageJ (NIH) by observers blinded to the genotype of the samples.
Vs1000s
Manufactured by Leica
Sourced in Germany
The VS1000s is a high-performance digital microscope system designed for advanced imaging and analysis in laboratory settings. It provides superior optical quality and advanced features to support a wide range of scientific applications.
Automatically generated - may contain errors
Lab products found in correlation
Diamond knife, by Electron Microscopy Sciences (2 mentions)
Jem 1230 electron microscope, by JEOL (2 mentions)
Em uc6, by Leica (2 mentions)
Hq silver kit, by Nanoprobes (1 mentions)
Eclipse ti2e confocal microscope, by Nikon (1 mentions)
Glur2 3, by Merck Group (1 mentions)
Cytochrome c type 3, by Merck Group (1 mentions)
5 protocols using vs1000s
1
Ultrastructural Analysis of CCR2 Immunoreactivity in Mouse Spinal Dorsal Horn
2
Quantification of Interneuron Loss After Epilepsy
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Two weeks after IHKA injection, animals were deeply anesthetized and transcardially perfused with ice-cold saline and then with 4% paraformaldehyde in 0.1 M PB (pH = 7.4). Brains and immersion fixed acute slices (patch clamp experiments) were cut into 60-μm-thick sections in the coronal plane with a vibratome (Leica, VS1000s). On selected sections, immunoreactivities were tested: SATB1 (mouse, 1:500, SantaCruz, sc-376096) and GluR 2/3 (rabbit, 1:500, Millipore, 07–598) were diluted in 0.1 M PB and incubated overnight at room temperature. For detection, fluorescent dye (Alexa488/Alexa594) conjugated donkey secondary antibodies (Jackson ImmunoResearch Labs, 715-545-150/715-585-150) raised against the host species of primary antibodies were applied on the sections. To determine MC loss after SE, 10 sections (60 μm) per mouse (every third section) were collected starting from AP: − 1.4 mm to AP: − 3 mm from the bregma and all SATB1 positive cells in the hilus were counted. In certain sections, immunohistochemistry was combined with RNAscope in situ hybridization as described previously [42 (link)]. Confocal images were taken with a Nikon Eclipse Ti2-E confocal microscope with 10x and 20x objectives.
3
Cytochrome Oxidase Histochemistry in Brainstem
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Four rats from each group were deeply anesthetized and perfused transcardially with 150 ml saline, followed by 50 ml ice-cold mixture of 4% paraformaldehyde and 0.05% glutaraldehyde in 0.1 M PB for 1 h. Brainstems were removed and postfixed by immersion in the same fixative for 4 h at 4mersion in the same fixative for μm thicknesses were prepared with a vibratome (VS1000s, Leica). Sections (n = 18–20) including the pre-BötC region were collected from each brainstem. The basic protocol for CO histochemistry was as described previously (Liu et al., 2001 (link)). Briefly, sections were incubated in 0.1 M PB containing 25 mg 3, 3’-diaminobenzidine (DAB, Sigma), 15 mg cytochrome c, type III (Sigma), and 2 g sucrose per 50 ml solution. They were incubated at 37 for 3 h in the dark. All sections from three groups were reacted together to avoid differences due to slight variations, such as temperature, medium composition, or incubation time. After CO histochemistry, sections were washed three times, 5 min for each in cold 0.1 M PB. As CO-negative control experiment, two NOR rats were perfused and sectioned as above, and performed SST and NK1R immunocytochemistry procedures, avoiding CO histochemistry.
4
Ultrastructural Analysis of Hippocampal CA1 Region
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Mice were anesthetized with 2% pentobarbital sodium (20 mg/kg) and transcardially perfused with 10 mL of saline, followed by 50 mL of ice‐cold mixture of 2% paraformaldehyde and 2% glutaraldehyde in 0.1 M phosphate buffer (PB) for 1 h. The brains were removed and postfixed by immersion in the same fixative for 4 h at 4°C. Serial coronal sections (50 μm thick) were prepared with a vibratome (VS1000s, Leica). The sections were postfixed in 1% osmium tetroxide in 0.1 M PB for 2 h. Then, the sections were dehydrated with graded ethanol, incubated with propylene oxide, and finally embedded in Epon 812 between plastic sheets. After polymerization, flat‐embedded sections were examined under a light microscope. The hippocampal CA1 region was selected, trimmed under a stereomicroscope, and then glued onto blank resin stubs. Serial ultrathin sections were cut with a diamond knife (Diatome, Hatfield, PA) and an ultramicrotome (Leica EM UC6, Wetzlar, Germany) and mounted on Formvar‐coated mesh grids (6–8 sections/grid). The sections were then counterstained with uranyl acetate and lead citrate for electron microscopic examination. The ultrathin sections were examined under a JEM‐1230 electron microscope (JEOL LTD, Tokyo, Japan) equipped with a CCD camera and its application software (832 SC1000, Gatan, Warrendale, PA).
5
Ultrastructural Analysis of Mouse mPFC
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The C57BL/6J mouse brains were fixed with 2% glutaraldehyde and 2% PFA for 2 h at room temperature. A vibratome (Leica, VS1000s, Germany) was utilized for preparing serial coronal sections at 50 μm thickness. Sections containing the mPFC region were collected, then rinsed by a PB buffer, and post-fixed with 1.0% osmium tetroxide for 2 h in a precooled cacodylate buffer. mPFC sections were dehydrated through an ethanol gradient from 30 to 100%, then transferred to propylene oxide, and finally embedded in Epon 812 between plastic sheets for 12 h at 60°C. These sections were cut using an ultramicrotome (Leica, EM UC6, Germany), collected on formvar-coated grids, and stained with uranyl acetate and lead citrate. Finally, they were examined using an electron microscope (JEOL LTD, 1230, Japan) at 80 kV. Images were obtained using an AMT digital imaging system (Gatan, 832 SC1000, United States).
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