For visualization of Neurobiotin, the sections were incubated for 2–3 h at room temperature with Cy2-or Cy3-conjugated streptavidin (1:1000; Jackson ImmunoResearch). When combined with immunohistochemistry (see below) the streptavidin was added to the secondary antibody and the sections incubated for 2–3 h. For immunohistochemical detection of tyrosine hydroxylase (TH), serotonin (5-HT) or GABA, sections were incubated overnight at 4 °C with a mouse anti-tyrosine hydroxylase antibody (1:500; MAB318; Millipore), a rabbit anti-serotonin antibody (1:1000; ImmunoStar, Inc.), or a mouse monoclonal anti-GABA antibody (1:5000, mAb 3A12; RRID AB_2314450 ; kindly donated by Prof. Peter Streit, Brain Research Institute, University of Zurich, Switzerland), respectively. Following a thorough rinse in phosphate buffered saline (PBS) the sections were incubated with donkey anti-mouse IgG-Cy3 or donkey anti-rabbit IgG-Cy3 (1:500; Jackson ImmunoResearch). Slides were then rinsed in PBS for 3 × 10 min and cover-slipped with glycerol containing 2.5% DABCO (Sigma-Aldrich). All antisera were diluted in 1% BSA and 0.3% Triton-X 100 in 0.1 M PB. In addition, all sections were counterstained with a fluorescent Nissl stain (1:1000; MolecularProbes).
Nissl stain
Manufactured by Thermo Fisher Scientific
Sourced in United States
Nissl stain is a histological staining technique used to visualize the structure and distribution of nerve cells in the central nervous system. It selectively stains the Nissl bodies, which are accumulations of rough endoplasmic reticulum within the cytoplasm of neurons. This staining allows for the identification and analysis of neuronal morphology and cytoarchitecture.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Mab318, by Merck Group (1 mentions)
Cy3 donkey anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Cy3 donkey anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Cy2 or cy3 conjugated streptavidin, by Jackson ImmunoResearch (1 mentions)
Dabco, by Merck Group (1 mentions)
Anti neun, by Merck Group (1 mentions)
Avertin, by Merck Group (1 mentions)
Anti gfap, by Agilent Technologies (1 mentions)
Vt1000, by Leica (1 mentions)
Neurotrace, by Thermo Fisher Scientific (1 mentions)
3 protocols using nissl stain
1
Immunohistochemical Visualization of Neurotransmitters
2
Histological Analysis of Mouse Brain
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After intraperitoneal injections of avertin (250 mg/kg, Sigma, St. Louis, MO, USA) mice were transcardially perfused with freshly prepared 4% paraformaldehyde in 0.1 mol/L cacodylate buffers, pH 7.4 and brains were removed and embedded in a gelatin matrix (NeuroScience Associates, Knoxville, TN, USA). Free-floating sections (35 μm in thickness) were cut on a microtome and processed for staining. Histological analyses with cresyl violet (Nissl stain, Molecular Probe, Eugene, OR, USA), amino-cupric staining, and immunohistochemistry against anti-NeuN (Chemicon, Temecula, CA, USA) and anti-GFAP (DAKO, Glostrup, Denmark) were performed as previously described [10 (link)]. The staining specificity of these antibodies was assessed by omission of the primary antibodies.
3
Visualizing Fenestrated Capillaries in Mice
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To evaluate whether capillaries from the SVCC are fenestrated, Evans blue solution (4% in physiologic saline solution) was administrated intraperitoneally (100 mg/kg) in 5 adult mice. When the skin of the mice turned blue (after 24 h), the mice were anesthetized and intracardially perfused with physiological saline solution. Brains were dissected immediately, and 50-μm slices were obtained using a vibratome (Leica, VT1000S). In addition, slices were labeled with the fluorescent marker for Nissl substance (Neuro Trace, Nissl stain, Molecular Probes, N-21480) and mounted. Samples were observed under a Zeiss LSM 780 confocal microscope, and 1.0-μm optical slices were obtained from 40-μm stacks.
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