Additionally, hSC-loaded scaffolds were used for cell specific immunocytochemical stainings. Therefore scaffolds were fixed for 30′ in 4% paraformaldehyde solution in 0.1 M phosphate buffer (PB, 4°C) and subsequently cut in 100 μm sections on a vibratome (Leica VT1000S3) and mounted on glass slides. Then sections were washed 3x with 0.1 M PBS and blocked with 3% normal goat serum (Sigma, Munich, Germany), 0.5% bovine serum albumin (BSA, fraction V), and 1% Triton X-100 in 0.1 M PBS for 60 min. Sections were then incubated overnight using the following primary antibodies: mouse monoclonal antineurofilament 200 kDa (NF200; clone NE14, 1 : 5000, Sigma), mouse monoclonal antivimentin (clone V9, 1 : 20,000, Sigma), or rabbit polyclonal anti-S100β (1 : 1000, Dako, Hamburg, Germany). Subsequently, fluorescent conjugated secondary antibodies (goat anti-rabbit, Alexa-488, and goat anti-mouse, Alexa-594, both 1 : 500, Molecular Probes, Paisley, UK) were incubated with primary antibodies 3 h at room temperature. Subsequently every consecutive section was counterstained using the nuclear dye 40,6-diamidino-2-phenylindole (DAPI, 1 mg/mL, 5 min). Negative controls were performed via omitting primary antibodies and revealed no non-specific immunostaining.
Anti s100β
Manufactured by Agilent Technologies
Sourced in Germany, Denmark, United States
Anti-S100β is a laboratory diagnostic product manufactured by Agilent Technologies. It is a specific antibody that targets the S100β protein, which is a biomarker for various neurological conditions.
Automatically generated - may contain errors
Lab products found in correlation
Alexa 594, by Thermo Fisher Scientific (1 mentions)
Normal goat serum (ngs), by Merck Group (1 mentions)
Mouse monoclonal anti vimentin clone v9, by Merck Group (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Anti iba1, by Biocare Medical (1 mentions)
Anti o4, by Merck Group (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Dnase 1, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Biosera (1 mentions)
Blocking solution, by Agilent Technologies (1 mentions)
Antibody diluent solution, by Agilent Technologies (1 mentions)
Target retrieval solution, by Agilent Technologies (1 mentions)
Anti mouse alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Anti gfap, by Agilent Technologies (1 mentions)
Anti epcam, by BioLegend (1 mentions)
Anti rabbit cy3, by Jackson ImmunoResearch (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
8 protocols using anti s100β
1
Immunocytochemical Analysis of hSC-Loaded Scaffolds
2
Isolation and Purification of Astrocytes
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Cortices were isolated from P21 Swiss Webster and collected in calcium/magnesium-free Hank’s balanced salt solution (HBSS), trypsinised and DNAseI treated (50 μg/ml, Sigma) for 20 min at 37 °C and mechanically dissociated into a homogenous cell suspension [28 (link)]. Following washes and centrifugation, mixed glial cells were plated onto PDL (Sigma) in DMEM/F12 (Invitrogen) with 100 U/ml penicillin/100 mg/ml streptomycin (Sigma) and 10 % FBS (Biosera). Once confluent, cultures were shaken overnight at 180 rpm to remove microglia and oligodendrocytes and treated for 4–7 days with 20 μM cytosine arabinoside (AraC) to kill remaining dividing cells and obtain essentially pure astrocytes (>98 %), determined by immunofluorescence using anti-glial fibrillary acidic protein (GFAP) (Millipore) and anti-S100β (Dako) for astrocytes, anti-Iba1 (Biocare, microglia), anti-O4 (Sigma, oligodendrocytes) and TuJ1 (Covance, neurons).
3
Immunofluorescence Staining of Brain Tissue
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Tissues were fixed in 4% paraformaldehyde overnight at 4 °C. Following paraffin-embedding and sectioning, slides were baked at 60 °C for 2 h, deparaffinized with successive incubations in xylene, absolute ethanol, 95% ethanol and 70% ethanol, and incubated in 1X Target Retrieval Solution (Dako) at 110 °C for 90 s. Sections were then successively incubated in blocking solution (Dako) for 1 h, primary and secondary antibodies diluted in antibody diluent solution (Dako) overnight at 4 °C and 1 h at room temperature, respectively. The following primary and secondary antibodies were used: rabbit polyclonal anti-S-100β (1:1000, DAKO), rabbit polyclonal anti-GFAP (1:500, DAKO), mouse polyclonal anti-EpCAM (1:200, Biolegend), anti-rabbit-Cy3 (1:500, Jackson) and anti-mouse-Alexa Fluor 488 (1:500, Invitrogen). Stained sections were imaged using an OLYMPUS BX51 microscope (objectives 20X FN 26.5, 40X FN 26.5) and with an OLYMPUS IX83 inverted microscope (objectives 20X FN 22, 40X FN 22).
4
Paraffin-Embedded Tissue Immunohistochemistry
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Tissue was embedded in paraffin, and 6-μm sections were cut, stained with either H&E or toluidine blue, and incubated overnight at 4°C with the following antibodies: anti-S100β (Dako, Carpinteria, CA), Ki67 (Novocastra Leica Microsystems, Buffalo Grove, IL), anti-BrdU, or anti-CC3 (Cell Signaling Technology, Danvers, MA). Visualization methods were as described (6 (link)).
5
Tissue Sectioning and Immunohistochemistry
6
Immunostaining of S100β and βIII-tubulin
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The following primary antibodies were used in this study: rabbit polyclonal anti-S100β (DAKO, 1:100, Cat# Z0311, immunostaining, RRID:AB_10013383), mouse anti-βIII-tubulin (Bio-legend, 1:500, Cat# 801203; immonostaining, clone# TUJ1; RRID: AB_2564757).
7
Visualizing Neurite Outgrowth and Schwann Cell Phenotype
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NG108-15 neuronal cells, and primary Schwann cells, were immunolabelled for specific proteins to visualize neurite outgrowth from neuronal cells and confirm Schwann cell phenotype, respectively, as previously described [24 (link), 27 ]. Briefly, samples were fixed with 3.7% (v/v) paraformaldehyde (20 min), permeabilized with 0.1% Triton X-100 (20 min) and blocked with 3% bovine serum albumin (BSA), in PBS (30 min) [24 (link), 27 ].
NG108-15 neuronal cells were labelled with a mouse anti-β III-tubulin antibody (1:250) (Promega, UK), for 48 h at 4°C, followed by a Texas Red-conjugated anti-mouse IgG antibody (1:200 dilution in 1% BSA from Vector Labs, USA), for 90 min at room temperature, to visualize and measure neurite outgrowth [24 (link), 27 ].
Schwann cells were identified with a polyclonal rabbit anti-S100β (1:250) (Dako, Denmark) antibody, incubated for 48 h at 4°C, followed by a FITC-conjugated secondary anti-rabbit IgG antibody (1:100 dilution in 1% BSA), for 90 min at room temperature [24 (link), 27 ]. All cells were incubated with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma Aldrich) (300 nM) for 30 min, at room temperature to observe cell nuclei [27 ]. Cells, and samples, were imaged using an upright Zeiss LSM 510 confocal microscope using three fields of view for quantitative analysis [24 (link), 27 ].
NG108-15 neuronal cells were labelled with a mouse anti-β III-tubulin antibody (1:250) (Promega, UK), for 48 h at 4°C, followed by a Texas Red-conjugated anti-mouse IgG antibody (1:200 dilution in 1% BSA from Vector Labs, USA), for 90 min at room temperature, to visualize and measure neurite outgrowth [24 (link), 27 ].
Schwann cells were identified with a polyclonal rabbit anti-S100β (1:250) (Dako, Denmark) antibody, incubated for 48 h at 4°C, followed by a FITC-conjugated secondary anti-rabbit IgG antibody (1:100 dilution in 1% BSA), for 90 min at room temperature [24 (link), 27 ]. All cells were incubated with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma Aldrich) (300 nM) for 30 min, at room temperature to observe cell nuclei [27 ]. Cells, and samples, were imaged using an upright Zeiss LSM 510 confocal microscope using three fields of view for quantitative analysis [24 (link), 27 ].
8
S100β Immunostaining and Electron Microscopy
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