NES cells were washed with PBS and fixed with 4% formaldehyde for 10 min at RT. After three additional washes in PBS, cells were left in blocking solution (PBS supplemented with 1% horse serum and 0.1% Triton) for 1 h at RT. Cells were incubated with primary antibodies o/n at 4 °C. Primary antibodies were diluted as follows: Nestin (1:200 R&D systems #MAB1259); PAX6 (1:200 BD Bioscience #561462); phospho-vimentin (1:200 Abcam #ab22651); SOX2 (1:400 Millipore #ab5603); vimentin (1:200 Abcam #ab16707); CHAT (1:500 Millipore #AB144P); Doublecortin (1:200 Cell Signaling #4604); GFAP (1:500 Sigma #G3893); RBFOX3 (1:500 Millipore #MAB377); TUBB3 (1:1000 Abcam #ab107216); Neurofilament (1:200 Millipore #MAB1615); MAP2 (1:1000 Millipore #AB5622); O4 (1:200 R&D systems #MAB1326) PSD95 (1:250 Thermo Fisher Scientific #51-6900); Synaptophysin (1:500 Millipore #MAB329); KI67 (1:500 Abcam #ab16667); HOXB9 (1:200 Abcam #ab66765). The following day, cells were washed in PBS three times before being incubated with Alexa 568 or Alexa 488 secondary antibodies (1:500 Invitrogen) for 1 h at RT. The samples were finally mounted using mounting medium (Life Technologies #P36935) containing DAPI.
G3893
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, Denmark, Macao
G3893 is a laboratory centrifuge designed for general-purpose applications. It features a robust construction and a simple user interface. The centrifuge can accommodate a variety of sample tubes and microplates, and it operates at adjustable speeds to meet the needs of different separation tasks. The product details and technical specifications are available upon request.
Automatically generated - may contain errors
Lab products found in correlation
Mab377, by Merck Group (17 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (8 mentions)
Axio observer z1, by Zeiss (6 mentions)
Ab5076, by Abcam (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
Ab97959, by Abcam (3 mentions)
Z0334, by Agilent Technologies (3 mentions)
Ab13970, by Abcam (3 mentions)
Ab18723, by Abcam (3 mentions)
Cbl1512, by Merck Group (3 mentions)
Alexa fluor 488 anti rabbit, by Thermo Fisher Scientific (3 mentions)
Mab5356, by Merck Group (3 mentions)
Ab15282, by Merck Group (3 mentions)
Anti mouse alexa fluor 555, by Thermo Fisher Scientific (3 mentions)
Tcs sp8 confocal laser scanning microscope, by Leica (3 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Mab1615, by Merck Group (2 mentions)
Alexa 568, by Thermo Fisher Scientific (2 mentions)
79 protocols using g3893
1
Immunostaining of Neural Stem Cells
2
Immunodetection of Cerebellar Cell Markers
3
Immunofluorescence Analysis of DRG, Spinal, and Skin Tissues
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Tissue sections (thickness, 5 µm) of the 4th and 5th lumbar DRG, spinal dorsal horn and innervated skin were dried and then incubated in blocking buffer containing 0.2% Triton X-100 and 1.5% normal goat serum in PBS at room temperature. Theses sections were washed twice with PBS, then incubated with the primary antibodies (TSLP (1:100; Sigma; PRS4025), TSLPR (1:500; Sigma; WH0064109M3), GFAP (1:200; Sigma; G3893), NeuN (1:400; Millipore; MAB377), iba1 (1:100; proteintech; 10904-1-AP), ATF3 (1:500, Sigma; HPA001562), PGP9.5 (1:200; proteintech; 14730-1-AP)) at 4 °C overnight, further washed three times with PBS, and replaced in secondary antibodies at room temperature for 2 h. Negative controls in which the primary antibody was omitted were conducted. All immunofluorescence staining was obtained in a minimum of 4–6 serial sections for each rat. Theses sections were photographed by using a fluorescence microscope (Olympus, Commonwealth of Pennsylvania, USA, U-RFL-T). The fluorescence intensity of GFAP were captured from 5–6 randomly chosen fields in each section (the medial superficial dorsal horn laminae I–III) and measured with Image J (National institutes of Health, Bethesda, MA, USA). The background intensity was subtracted in each section and the intensity was presented as fold increase compared to the control.
4
Immunohistochemical analysis of myelin and neuroinflammation
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Sagittal 8 μm sections were cut on a Leica CM1960 cryostat. Antigen retrieval was performed using the 2100 Retriever and Diva decloaker buffer as described by the manufacturer (Dako, Glostrup, Denmark), unless otherwise specified. Antibodies used: Iba1 (1:1000, Wako chemicals 019-19741), GFAP (1:1000, Sigma G3893), NOGO-A (1:500, Millipore AB5664P), β-APP (1:1000, Abcam ab32136), PLP1 (1:1000, AbD Serotec MCA839G), MBP (1:500, without antigen retrieval, Abcam ab24567), and neurofilament (1:1000, Millipore MAB1615). Secondary antibodies were Alexa Fluor 488 and 594 anti-mouse or anti-rabbit. Pictures were taken with a Nikon TE2000, with a 10 × or 40× objective, or a Leica Confocal SP2 with 40× or 63× objective. Myelin was analysed by visual scoring of demyelination on a scale from 0 (no demyelination) to 3 (total demyelination), as previously described (Skripuletz et al., 2010; Wergeland et al., 2011) . Results are given as a mean between the score for PLP1 and MBP. For cell number analysis, numbers are given as a mean from 2 pictures within the subcortical region and 2 pictures from the cerebellar cortex (Fig. 1b). β-APP was measured by counting particles in the range of 10-600 pixels using the FIJI software. 2-4 sections were analysed for each animal per antibody. All analyses were done blinded.
5
Immunocytochemistry of Glial Cells
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Primary antibodies (GFAP monoclonal mouse antibody, Sigma-Aldrich G3893, 1:1,000; rabbit OLIG2, Chemicon AB9610, 1:300) were incubated overnight at 4°C. O4 staining was performed on live cells (O4 hybridoma). Goat secondary antibodies conjugated to Alexa dyes (Molecular Probes) were added at 1:1,000 for 1 hr at room temperature and DAPI was used as a nuclear counterstain.
6
Immunostaining of Neural Stem Cell Markers
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Primary antibodies (GFAP monoclonal mouse antibody, Sigma-Aldrich G3893 1:1000; rabbit OLIG2, Chemicon AB9610, 1:300; mouse monoclonal nestin, R&D MAB1259, 1:500; rabbit SOX2, Abcam ab97959; 1:1000; rabbit MAP2, Abcam ab32454, 1:1000; rabbit vimentin, Abcam ab45939, 1:900), were incubated overnight at 4°C. Goat secondary antibodies conjugated to Alexa dyes (Molecular Probes), 1:1000, were added for 1 hr at room temperature and DAPI was used as a nuclear counterstain. For tumor spheres; 10-12 μm thick sections of embedded spheres were cut with a Leica CM3050S Cryostat and mounted on Superfrost™ Plus Slides (Fisher Scientific), which were stored in -20°C until staining and imaging as described above. The Operetta (Perkin Elmer) was used for acquisition of images and the Harmony software for quantification of cells.
7
Immunocytochemistry of Primary Hippocampal Cultures
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To verify the cellular content, primary hippocampal cultures were subjected to immunocytochemical staining on day 21 of culture development in vitro (DIV). The cultures were fixed with 4% paraformaldehyde for 15 min at room temperature, followed by incubation with a solution of 0.2% Triton X-100/PBS for effective cell permeabilization. For immunofluorescence reactions, the cultures were then incubated for 2 h in the presence of a polyclonal mouse anti-GFAP (glial fibrillary acidic protein, a marker of differentiated astrocytes) primary antibody (Sigma-Aldrich G3893, Merck KGaA, Darmstadt, Germany, 1:500 dilution) and polyclonal rabbit anti MAP2 (a marker of differentiated neurons) primary antibody (Abcam 32454, Cambridge, UK, 1:500 dilution). Next, the cultures were subjected to a 45-min incubation in the following secondary antibody mixture: goat anti-mouse Alexa 647 (Thermo Fisher Scientific, A21236, Waltham, MA, USA, 1:800 dilution) and chicken anti-Rabbit Alexa Fluor 488 (Thermo Fisher Scientific, A21441, Waltham, MA, USA, 1:800 dilution). The stained material was observed using a Zeiss LSM 800 fluorescence confocal microscope (Carl Zeiss, Oberkochen, Germany).
8
Immunocytochemical staining of astrocytes
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Immunocytochemical staining of astrocytes was performed as described previously (Stenovec et al., 2016 (link)). The following primary and secondary antibodies were used: mouse monoclonal anti‐nestin (dilution 1:400; ab11306, Abcam), mouse monoclonal anti‐GFAP (dilution 1:500; G3893, Merck), rabbit polyclonal anti‐MFGE8 (dilution 1:250; sc33546, Santa Cruz Biotechnology, Dallas, TX, USA), and anti‐mouse or anti‐rabbit antibodies conjugated to Alexa Fluor 488 or 546 (1:600; Thermo Fisher Scientific). The counts of immunolabeled GFAP‐ and/or MFGE8‐positive astrocytes were obtained as described in the section on Astrocyte viability and analysis.
9
Immunohistochemical Labeling of Microglia and Astrocytes
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For immunohistochemical labeling, slices in 24-well plates were incubated overnight at +4°C with Rabbit polyclonal anti-Iba-1 (1:250, 019-19 741, Wako) and mouse monoclonal anti-GFAP (1:400, G3893, Sigma-Aldrich). Then, slices were incubated 1 hour at room temperature with secondary anti-rabbit Cy3 (1:1000) and secondary anti-mouse 488 (1:1000) antibodies. Slices were incubated 5 minutes with DAPI 10 μg/mL and rinsed with phosphate buffer saline. Finally, slices were mounted with ProLong (ThermoFischer). Pictures of each slices were taken with a confocal Microscope (Leica SPE).
10
Quantitative Immunohistochemical Analysis of Dopaminergic Neurons
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Mice were anesthetized and perfused with 4% paraformaldehyde. Brains were extracted, dehydrated in 30% sucrose solution, embedded in OCT, and continuously sliced at 25 μm thickness. The immunohistochemistry staining was processed as previously described31 (link): tissue sections were blocked with 3% H2O2 for 15 min and 5% goat serum for 30 min at room temperature after heat-induced antigen retrieval. The sections were then incubated with primary antibody to TH (1:500, AB152, Merck-Millipore) at 4 °C for 12 h and followed by incubating with horseradish peroxidase (HRP)-labeled goat anti-rabbit (1:300; 31460, ThermoFisher Scientific). The number of TH-positive cells in the SN area was determined by stereological methods, as previously described32 (link). For immunofluorescence staining: tissue sections were blocked with 5% goat serum for 30 min at room temperature and were incubated with primary antibodies to IBA1 (1:500, 019-19741, Wako) and GFAP (1:500, G3893, Sigma–Aldrich) at 4 °C for 12 h. The sections were then incubated with fluorescent-labeled goat anti-rabbit (1:500, 111-545-144, Jackson ImmunoResearch) and goat anti-mouse (1:500, 115-165-075, Jackson ImmunoResearch). The related fluorescence intensities were analyzed by image pro plus software.
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