Slides were incubated with the following primary antibodies at 4°C overnight (8 hours): rabbit anti-ionized calcium binding adapter molecule 1 (Iba-1; 1:500; Abcam, Cambridge, UK, Cat# ab178846; RRID: AB_2636859), as Iba-1 is up-regulated in microglia following nerve injury (Wu et al., 2022); mouse anti-glial fibrillary acidic protein (GFAP; 1:300; Cell Signaling Technology, Danvers, MA, USA, Cat# 3670S; RRID: AB_561049), as GFAP is expressed in the central nervous system in astrocytes (Kisucká et al., 2021); and rabbit anti-neurofilament-200 (NF200; 1:2000; Cell Signaling Technology, Cat# 2836S; RRID: AB_10694081), as NF200 is expressed in the central nervous system in neurofilaments (Xu et al., 2015). Next, the slides were incubated with fluorescent Alexa Fluor488 goat anti-mouse (1:1000; Invitrogen, Carlsbad, CA, USA, Cat# A32723TR; RRID: AB_2866489) and Alexa Fluor594 goat anti-rabbit secondary antibodies (1:1000, Invitrogen, Cat# A32740; RRID: AB_2762824) for 60 minutes at 37°C. An inverted fluorescence microscope (Leica DM6000, Wetzlar, Germany) was used to capture images and conduct further analysis. To determine the total area of neurofilament protein, the NF200-positive area in spinal cord was calculated. Iba-1 and GFAP-positive areas were observed to assess aggregation of microglia and astrocytes, respectively (Zeng et al., 2019).
Nf 200
Manufactured by Cell Signaling Technology
Sourced in United States
The NF-200 is a laboratory instrument that measures the electrical signal propagation in neuronal cells. It is used to study the properties of nerve fibers and their function in the nervous system.
Automatically generated - may contain errors
Lab products found in correlation
Glial fibrillary acidic protein (gfap), by Cell Signaling Technology (2 mentions)
Gap43, by Cell Signaling Technology (2 mentions)
Lsm 510 meta confocal microscope, by Zeiss (2 mentions)
Iba 1, by Abcam (1 mentions)
Alexa fluor594 goat anti, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Dm6000, by Leica (1 mentions)
Ab6939, by Abcam (1 mentions)
Neuronal nuclei (neun), by Merck Group (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Cm1850, by Leica (1 mentions)
Neuronal nuclei (neun), by Cell Signaling Technology (1 mentions)
β tubulin, by Santa Cruz Biotechnology (1 mentions)
5 protocols using nf 200
1
Quantifying Neuroinflammatory Changes after Injury
2
Spinal Neurofilament Immunohistochemistry
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The longitudinal sections of the spinal tissues were collected from rats on the 6th week. The sections were fixed in 40 g/L paraformaldehyde for 15 min, washed in phosphate buffer saline (PBS), and blocked in PBS containing 0.1% Triton X-100 and 5% bovine serum albumin (BSA) at 37 °C for 50 min. Next, the slides were incubated with neurofilament 200 antibody (NF-200, 1:200, #55453, Cell Signaling Technology (CST), Beverly, MA, USA) at 4 °C overnight and then with cy3-labeled secondary antibody goat anti-rabbit immunoglobulin G (IgG) (ab6939, 1:500, Abcam Inc., Cambridge, MA, USA) at room temperature for 60 min. Then, the slides were sealed and observed under a LSM510 meta confocal microscope (Carl Zeiss MicroImaging, Inc., Thornwood, NY, USA). The gray matter at the region 2–3 mm posterior to the spinal injury site was used for measurement. Relative NF-200-positive area was calculated using an Image J 2.0 software.
3
Protein Expression Analysis in Cells
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The cultured cells are washed 3 times with cold Phosphate Balanced Solution (PBS). Then the Radio-Immunoprecipitation Assay (RIPA) lysis buffer is added to the cells at 4°C for 10 minutes. The mixture is centrifuged at 12,000 rpm, 4°C for 10 minutes. 10% Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) is used to separate the equal amounts of the protein. When the proteins are transferred to the Polyvinylidene Fluoride (PVDF) membrane, the 5% BSA is used to bind the protein of the membrane for 1 h at room temperature. Subsequently, the membrane is incubated with primary antibodies which are the PTEN, NF-200, and GAP-43 (Cell Signal Technology, BSN, USA) at 4°C overnight. After washing 3 times with the PBS, a goat-anti-rabbit IgG-HRR secondary antibody (Thermo Pierce, MA, USA) is used for incubating the membrane at room temperature for 2 h. Protein levels are normalized to GAPDH by using a mouse monoclonal anti-GAPDH antibody (Thermo Pierce, MA, USA).
4
Tracking hADSC-NCs in Mouse Brain
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The EGFP-hADSC-NCs were tracked and identified by immunohistochemical staining with the neuron markers MAP2 (Synaptic Systems, Cat#188011 and Cat#188003), NF-200 (Cell Signaling, Cat# 2836), NeuN (Sigma, Cat# SAB4300883), Synapsin1/2 (Cell Signaling, Cat#5297) Human Nuclear Antigen (HuNA, Merck Millipore, Cat# MAB1281), the proliferative marker Ki67 (R&D Systems, Cat#AF7649), astrocyte marker of GFAP (Synaptic Systems, Cat#173011), microglia marker of IbaI (FUJI FILM, Cat# 019-19741), the cell apoptosis marker Caspase 3 (Cell Signaling, Cat# 9579S) and blood vessel endothelial marker of CD31(R&D Systems, Cat#AF3628) by following previously published methods [72 (link)]. Briefly, after the Morris water maze test, mice from each group were sacrificed, and intracardial perfusion was performed. Then, the brains were removed, fixed with 4% paraformaldehyde and dehydrated with a sucrose gradient. Brain sections 20 μm in thickness were obtained with a cryostat (Leica, CM1850), followed by immunohistochemical staining. The distribution of the EGFP-labeled hADSC-NCs in specific brain sites was observed through confocal microscopy (Leica, SP8). Additionally, colocalization was carefully observed to determine whether any transdifferentiation of hADSC-NCs into neuronal cells had occurred.
5
Plasmid Construction and Antibody Catalogue
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pEGFP-N1-Ngb wild-type (WT) and mutants (E53Q, E118Q, K119N, H64A, H64L, Y44D)19 (link) and pGenesil-1-shNgb (short hairpin Ngb)20 (link) were previously constructed. pEGFP-C1-Ngb WT and truncates (Δ1–6, Δ1–18, Δ1–35, Δ1–42, Δ106–151, Δ123–151, Δ128–151), pDEST26-CV-p38, and pDEST-Flag-NV-p38 were constructed in our laboratory. EST26-CV (C-terminal 137–238 Venus), pDEST26-CV-p38, pDEST-Flag-NV (N-terminal 1–157 Venus), pDEST-Flag-NV-p38, pDEST27-GST, and pDEST27-GST-p38 were kindly provided by Professor Haian Fu (Department of Pharmacology, School of Medicine, Emory University, USA). pDEST26-CV-Ngb and pDEST-Flag-NV-Ngb were previously constructed18 (link). p-FU-p38K53A mutant plasmid was constructed by PCR amplifying p38K53A fragment (with stop codon) using specific primers and pDEST-Flag-NV-p38 template and then cloning into p-FU-Venus plasmids at BamH1/BsrG1sites. All plasmids were confirmed by sequencing before use. Antibodies against Ngb, Flag (Sigma, Saint Louis, MO, USA), GAP43, GFAP, NeuN, NF200, p-p38, p38 (Cell Signaling Technology, Boston, USA), β-tubulin, green fluorescent protein (GFP), Cygb, GST, His (Santa Cruz Biotechnology, Santa Cruz, USA), and Tau-1 (Merk Millipore Ltd., Darmstadt, Germany) were purchased.
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