After the mice were transcardially fixed with 4% paraformaldehyde, the spinal cord was removed, dehydrated and embedded in OCT compound. The frozen tissue was cut in the sagittal or axial plane in 16 μm sections. For immunostaining, the sections were stained overnight at 4 °C with primary antibodies against MBP (myelin marker, 1:200, rat, Chemicon), NF200 (neuronal fiber marker, 1:200, rabbit, Sigma-Aldrich), NF200 (1:200, mouse, Sigma-Aldrich), 5-HT (serotonergic marker, 1:200, goat, ImmunoStar), VGluT2 (presynaptic marker, 1:200, guinea pig, Synaptic Systems), PSD95 (postsynaptic marker, 1:200, rabbit, Invitrogen), Hu (neuronal marker, 1:1000, human, a gift from Dr. Robert Darnell, The Rockefeller University, New York, NY, USA), Bassoon (pan-presynaptic marker, 1:200, mouse, Stressgen), NeuN (neuronal marker, 1:1000, mouse, Chemicon), c-fos (neuronal activity marker, 1:200, rabbit, Santa Cruz Biotechnology), or ChAT (1:100, goat, Chemicon). Then the sections were then incubated at room temperature for 1 h with Alexa Fluor-conjugated secondary antibodies (1:200, Invitrogen). Nuclear counterstaining was obtained using Hoechst 33342 (Molecular Probes).
Nf200
Manufactured by Merck Group
Sourced in United States
The NF200 is a laboratory equipment product manufactured by Merck Group. It is designed to perform nanofiltration, a separation process used to remove small molecules and ions from solutions. The NF200 utilizes a semi-permeable membrane to selectively filter and concentrate desired components in the solution. Its core function is to enable efficient and controlled separation of substances within a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Neuronal nuclei (neun), by Merck Group (9 mentions)
Glial fibrillary acidic protein (gfap), by Merck Group (7 mentions)
Glial fibrillary acidic protein (gfap), by Agilent Technologies (5 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (4 mentions)
Lsm 700, by Zeiss (4 mentions)
Vglut1, by Synaptic Systems (3 mentions)
Nestin, by R&D Systems (3 mentions)
β 3 tubulin, by R&D Systems (3 mentions)
Alexa fluor 633 goat anti rabbit igg h l, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 488 goat anti mouse igg h l, by Thermo Fisher Scientific (3 mentions)
A1 laser scanning confocal microscope, by Nikon (3 mentions)
Donkey anti rabbit secondary antibody, by Thermo Fisher Scientific (2 mentions)
Lsm200, by Zeiss (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Bio-Synthesis (2 mentions)
Primary antibodies against fibronectin, by Santa Cruz Biotechnology (2 mentions)
Cm1850, by Leica (2 mentions)
Cryostat, by Leica (2 mentions)
Iba 1, by Abcam (2 mentions)
Macro h2a 1, by Abcam (2 mentions)
Ctip2, by Abcam (2 mentions)
55 protocols using nf200
1
Immunohistochemical Analysis of Spinal Cord
2
Evaluating Cochlear Hair Cell Toxicity
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To evaluate the toxicity of H2O2, cultured organ of Corti explants were immunolabeled with a rabbit polyclonal antibody against myosin 7A (1/300, Proteus Biosciences Inc. #25-6790) and a mouse monoclonal antibody against neurofilament (NF 200, 1/600, Sigma-Aldrich #N0142) to label hair cells and spiral ganglion neurons, respectively. Hair cell counting (six cochleae per condition and per time point) was performed using standard techniques [20 (link)]. Due to the resistance of apical turn hair cells to H2O2 cytotoxicity, counting concerned only a 1.5-mm length of the cochlear duct at the basal turn (4 to 5.5 mm from the apex, see [12 (link)]).
The TUNEL kit (DeadEnd™ fluorometric TUNEL System, Promega #G3250) was used to identify apoptotic DNA fragmentation. The cochlear samples were counterstained with a rabbit polyclonal antibody against myosin 7A and a mouse monoclonal antibody against neurofilament. All secondary antibodies were used at a dilution of 1/1000. This included donkey anti-mouse and anti-rabbit IgG conjugated to Alexa 488 or Alexa 568 (Molecular Probes #A-21202, #A-21206, #A-10037, #A-10042). Fluorescent tags were visualized using a confocal microscope (LSM 5 Live Duo, Zeiss). In control specimens without primary antibodies, neither Alexa 488 nor 568 fluorescent tags were observed. All experiments were performed in triplicate.
The TUNEL kit (DeadEnd™ fluorometric TUNEL System, Promega #G3250) was used to identify apoptotic DNA fragmentation. The cochlear samples were counterstained with a rabbit polyclonal antibody against myosin 7A and a mouse monoclonal antibody against neurofilament. All secondary antibodies were used at a dilution of 1/1000. This included donkey anti-mouse and anti-rabbit IgG conjugated to Alexa 488 or Alexa 568 (Molecular Probes #A-21202, #A-21206, #A-10037, #A-10042). Fluorescent tags were visualized using a confocal microscope (LSM 5 Live Duo, Zeiss). In control specimens without primary antibodies, neither Alexa 488 nor 568 fluorescent tags were observed. All experiments were performed in triplicate.
3
Immunofluorescence Staining of DRG Neurons
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DRG neurons in tissue sections and cultured cells were subjected to immunofluorescence staining as described in our previous study [20 (link)]. Primary antibodies used were polyclonal antibodies for Cdk5 (1:400; Santa Cruz Biotech.), p35 (1:1,000; Santa Cruz Biotech.), pSTAT3 (Y705) (1:100; Cell Signaling Technology), pSTAT3 (S7275; Cell Signaling Technology) (1:100), and NF-200 (1:1,000; Sigma-Aldrich Chemical Co.) and monoclonal antibody for NF-200 (1:400; Sigma-Aldrich Chemical Co.). Fuorescein-goat anti-mouse (1:400; Molecular Probes, Eugene, OR, USA) and rhodamine-goat anti-rabbit (1:400; Molecular Probes) secondary antibodies were used in this study.
4
DRG Neuron Culture and Analysis
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Primary DRG neurons were cultured as previously described (de Luca et al., 2015). Briefly, DRGs were collected from newborn 12-hour-old Sprague-Dawley rats, digested twice in 0.125% (w/v) collagenase type IV (Sigma) for 1 hour, continually trypsinized using 0.25% (w/v) trypsin for 30 minutes, and mechanically dissociated in DMEM/F12 (Gibco) supplemented with 1% N2 (Invitrogen) and 1% penicillin/streptomycin (Sigma). After centrifugation and resuspension, the cells were plated on non-coated (control group) or ECM-coated dishes (ECM group) at 5 × 104/cm2. After 72 hours of incubation, the cells were fixed in 4% paraformaldehyde for 15 minutes and allowed to incubate with polyclonal antibody against neurofilament heavy (NF200; 1:200; Sigma) for 12 hours at –4°C, followed by incubation with secondary antibody (Alexa Fluor®594) for 1 hour in the dark at 37°C. Images were collected by fluorescence microscopy (Olympus). The neurite length of DRG neurons was calculated using Image Pro Plus 5.0 software (Media Cybernetics) from five random fields of three independent samples.
5
Nerve Graft Evaluation via Immunofluorescence
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At specified time points after nerve grafting, the artificial nerve bridging effects were evaluated by observing the graft appearance. Grafts were frozen (-80°C), sectioned (6 µm thickness) using a cryostat (CM1850, LEICA, Germany), stained using immunofluorescent markers or HE, and examined under a laser scanning confocal microscope (LSM200, Zeiss, Germany) to identify different cell types and fibroin fibers within the graft. Nuclei were stained blue using 4’,6-diamidino-2-phenylindole (Biosynthesis Biotechnology, China). Primary antibody for low-affinity nerve growth factor receptor (NGFR) P75 (goat anti-rat; Santa Cruz Biotechnology, USA) was used to identify Schwann cells. Primary antibodies against fibronectin (goat anti-rat; Santa Cruz Biotechnology) and NF200 (rabbit anti-rat; Sigma, USA) were used to identify fibroblasts and axons, respectively. Schwann cells and fibroblasts were labelled green using donkey anti-goat secondary antibody and axons were marked red using donkey anti-rabbit secondary antibody (Life Technologies, USA). Integrin-α(rabbit anti-rat; Santa Cruz Biotechnology, USA ) were used to identify inflammatory cells. Specific steps for immunofluorescence staining were performed according to the manufacturer’s instructions. Some of the grafts were observed with a scanning electron microscope (SEM) (JSM-6510LV, Japan).
6
Immunofluorescence Staining of Cortical Neurons
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Cells cultured on coverslips were washed, fixed in 4% PFA for 10 minutes, and permeabilised in 0.4% Triton in PBS for 10 minutes. Coverslips were then blocked in 5% milk-PBS for 30 minutes and anti-HA (Sigma, 1∶1000) or NF-200 (Sigma, 1∶200) was incubated overnight at 4°C. Cells were then washed and incubated in secondary fluorescent-conjugated antibody (1∶1000) (Molecular probes, Invitrogen) for 2 hours and mounted in DAPI-containing Vectorshield mounting media (Vectors lab). Axons, visualized by NF-200 staining, were measured using ImageJ software (NIH). A minimum of 15 cortical neurons was quantified. TUNEL staining for apoptotic cells was carried out as per the manufacturer’s protocol (Roche, UK). Confocal LSM510 (Zeiss) or fluorescence (Leica) microscopes were used to acquire images using Axiovision software (Zeiss).
7
Immunohistochemical Analysis of Mouse Cochlea
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The temporal bones from the control and mutant mice were isolated and fixed for 1 h at room temperature or overnight at 4°C in 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA), followed by three washes with phosphate buffered saline (PBS). Finely dissected cochlear coils were permeabilized and blocked in 5% BSA, 2% normal goat serum and 0.1% Triton X-100 in PBS for 1 h. The tissue samples were washed and probed with primary antibody overnight at 4°C. We used the following primary antibodies in our studies: β-galactosidase (1:1000 dilution; MP Biomedicals, Solon, OH), myosin VIIa (1:200; Proteus BioSciences, Ramona, CA), synaptophysin (1:200; Abcam, Cambridge, MA), NF-200 (1:200; Sigma-Aldrich), Prox1 (1:100; Millipore, Billerica, MA) and Pericentrin (1:200; Millipore, Billerica, MA). After three washes with PBS, samples were probed with a fluorescently labeled Alexa-Fluor-488 or -546 secondary antibody (1:500; Life Technologies, Grand Island, NY) for 1 h at room temperature. Rhodamine-phalloidin or Alexa-Fluor-647-conjugated phalloidin (1:250; Life Technologies, Grand Island, NY) was used to label actin. Samples were mounted using ProLongGold (Life Technologies, Grand Island, NY) and viewed under an LSM 700 confocal microscope (Zeiss Microimaging Inc., Thornwood, NY) using a ×63, 1.4 N.A. oil-immersion lens.
8
Immunohistochemical analysis of DRG
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Perfusion was immediately performed through the ascending aorta with 4% paraformaldehyde after being anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg/kg). The DRG segments were removed and placed into the 4% paraformaldehyde for post-fixing overnight. Cryostat sections (16 μm) were cut, and immunohistochemistry was performed with primary antibody for Nav1.6 (1:100; Alomone labs), TNF-α (1:100, Santa Cruz), STAT3 (1:100; Abcam), phosphorylated STAT3 (Tyr705) (1:100; Abcam), IB4 (1:50; Sigma), GFAP (1:400; Chemicon), and NF-200 (1:200; Sigma). After incubation overnight at 4 °C, the sections were incubated with secondary antibodies, which conjugated with cy3 or FITC for 1 h at RT. To test the specificity of the TNF-α antibody, preincubation with TNF-α peptide was used [19 (link)]. Briefly, the TNF-α antibody was preincubated with TNF-α (R&D Systems, Inc.) in a concentration of 10 μg/mL, which was fivefold higher than that of anti-TNF-α antibody, in 4 °C for 1 h, and then the preincubated antibody was used for immunohistochemistry. The sections were examined with a Leica fluorescence microscope (Leica, Germany), and images were captured with a Leica DFC350 FX camera.
9
Immunohistochemical Profiling of Neural Markers
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Purchased reagents are indicated with their companies in brackets: monoclonal mouse antibody against neurofilament (NF-200, 1:1,000 Sigma-Aldrich, St. Louis, MO), monoclonal mouse antibody against vesicular GABA transporter (VGAT, 1:1,000; Synaptic Systems, Göttingen, Germany), polyclonal rabbit antibody against glial fibrillary acidic protein (GFAP, 1:1,000; Dako, Hamburg, Germany), polyclonal rabbit anti-ionized calcium-binding adapter molecule 1 (Iba-1; 1:1,000; Wako Chemicals, Neuss, Germany), polyclonal rabbit antibody against tyrosine hydroxylase (TH, 1:800; Chemicon, Hofheim, Germany), polyclonal rabbit antibody against vesicular glutamate transporter 1 (VGLUT1, 1:1,000; Synaptic Systems), polyclonal goat antibody against choline acetyltransferase (ChAT, 1:100; Merck Millipore, Darmstadt, Germany), secondary antibodies coupled with Cy2 or Cy3 (Jackson ImmunoResearch, Newmarket, UK).
10
Immunohistochemical Analysis of DRG Neurons
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Rats were deeply anesthetized, and the L4-6 lumbar DRG were removed quickly from the spinal cord, fixed with 4% paraformaldehyde in 0.1 M phosphate buffer overnight at 4°C. After the tissues embedded by paraffin, a series of 5 μm thick sections were cut for immunohistochemistry. Primary antibodies targeting rat NaV1.8 (1:200; Alomone), NaV1.9 (1:200; Alomone) and NF200 (1:200; Sigma) were performed for immunohistochemistry overnight at 4°C. After washing three times with PBST, the sections were incubated with secondary antibodies (1:100; sigma) for 1 h at 37°C. Fluorescent images were captured in an Olympus BX40 microscope (Olympus, Tokyo, Japan).
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