Immunolabelling was performed as described (Vieira et al., 2007 (link)) using rabbit anti-mouse TUJ1 (1:250; MRB-435P, BioLegend) and mouse anti-rat ISL1 (1:100; 39.4D5, DSHB) followed by Alexa Fluor 594-conjugated rabbit anti-goat Fab (1:200; A21223, Thermo Fisher) and Alexa Fluor 488-conjugated rabbit anti-mouse Fab (1:200; A11017, Thermo Fisher) or horseradish-peroxidase-conjugated goat anti-rabbit antibody (1:200; P0448, DAKO). To detect blood vessels, we used biotinylated isolectin B4 (1:500; L2140, Sigma) followed by Alexa Fluor 633-conjugated streptavidin (1:200; S-21375, Thermo Fisher). We used digoxigenin-labelled RNA probes for Isl1 (Schwarz et al., 2004 (link)), Hoxb1 (Gavalas et al., 2003 (link)), Hs6st1, Hs6st2 (Sedita et al., 2004 (link)), Erm (Trokovic et al., 2005 (link)) and Fgfr1-4 (Pringle et al., 2003 (link); Trokovic et al., 2003 (link)).
Mrb 435p
Manufactured by BioLegend
Sourced in United States
The MRB-435P is a laboratory equipment product. It is designed to perform a core function, but the details of its intended use are not included in this description.
Automatically generated - may contain errors
Lab products found in correlation
P0448, by Agilent Technologies (1 mentions)
L2140, by Merck Group (1 mentions)
S21375, by Thermo Fisher Scientific (1 mentions)
A11017, by Thermo Fisher Scientific (1 mentions)
Mo15012, by Neuromics (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Mab3418, by Merck Group (1 mentions)
Z0334, by Agilent Technologies (1 mentions)
Hoechst 33258, by Merck Group (1 mentions)
Mab65591, by R&D Systems (1 mentions)
Ix71 wide field microscope, by Olympus (1 mentions)
Mab1259, by R&D Systems (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Ac 74, by Merck Group (1 mentions)
Anti gfp, by Merck Group (1 mentions)
Cc117, by Merck Group (1 mentions)
Ab58742, by Abcam (1 mentions)
Fluorometric hdac6 activity assay kit, by Abcam (1 mentions)
A2052, by Merck Group (1 mentions)
Fm4 64fx, by Thermo Fisher Scientific (1 mentions)
6 protocols using mrb 435p
1
Immunofluorescence and in situ hybridization
2
Neuronal Cell Lineage Characterization
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Neuronal stem cells and differentiated neurons were fixed with 4% paraformaldehyde and probed using antibodies to nestin (MO15012, Neuromics Inc., Minneapolis, MN USA), microtubule-associated protein 2A (MAP2A, mab 3418, Millipore, Billerica, MA, USA), β-3 tubulin (Tuj1, MRB-435P, Biolegend, San Diego, CA, USA), and doublecortin (DCX, sc-8066, Santa Cruz Biotechnology, Dallas, TX, USA). Nuclear DNA was visualized by staining with 4′,6-diamidino-2-phenylindole (DAPI, ThermoFisher Scientific).
3
Immunofluorescence Assay of Neural Cell Markers
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BTICs were plated on poly-l -ornithine coated glass coverslips and induced to differentiate in serum free media supplemented with 1% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) for 5–7 days. For immunofluorescence assays, cells were fixed with 4% formaldehyde, permeabilized with 0.5% Triton X-100 and then incubated with the primary antibodies against Nestin (1: 800; MAB-1259, R&D Systems), Tubb3 (1:400; MRB-435P, Biolegend, San Diego, CA, USA), GFAP (1:200; Z0334, Dako, Glostrup, Denmark) or FSHR (1:30, MAB65591, R&D Systems) at 4 °C overnight as described.93 (link) After incubation with fluorophore-conjugated secondary antibodies (Invitrogen) and Hoechst 33258 (Sigma, St Louis, MO, USA) at 37 °C, coverslips were examined under an Olympus IX71 wide-field microscope. At least three independent experiments were performed and representative fields are shown in the immunofluorescence results.
4
Regulation of Microtubule Acetylation in Cells
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The following antibodies were used: CSPGs (2 μg/ml; CC117, EMD Millipore), cycloheximide (10 μg/ml; C0934, Sigma Aldrich), recombinant rat myelin-associated glycoprotein (MAG; 30 μg/ml; P07722, R&D Systems), Y-27632 ROCK inhibitor (10 μM; 1254, Tocris Bioscience), anti- αTAT1 (1:200; ab58742, Abcam), anti-HDAC6 (1:500; NB100-91805, Novus Biologicals), anti-acetylated α-tubulin (1:1000; D20G3, Cell Signaling Technology), anti-α-tubulin (1:5000; DM1A, Sigma-Aldrich), anti-β-actin (1:5000; AC-74, Sigma-Aldrich), anti-β III tubulin (1:5000; MRB435P, BioLegend) and anti-GFP (1:500; Sigma-Aldrich). Lentivirus containing GFP (control) or GFP-tagged wild-type αTAT1 constructs, under the human cytomegalovirus (CMV) promoter, was purchased from Dr. Mingjie Li (Washington University School of Medicine, St. Louis, MO; Li et al., 2010 (link)). HDAC6 activity was determined using the fluorometric HDAC6 Activity Assay kit (BioVision), as per manufacturer’s instructions.
5
Immunocytochemical Staining of Cell Cultures
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Cell cultures were fixed in 4% paraformaldehyde (100503, VWR) in PBS at room temperature (RT) for 20 min, then blocked for 1 hour in 10% normal goat serum (GS) (G9023, Sigma Aldrich) and 0.1% Triton X-100 (X100, Sigma Aldrich) in PBS. Cells were incubated with primary antibodies for 3 hours at RT. Secondary antibodies were applied for 1 hour in blocking buffer at RT. The primary antibodies used in this study were raised against rabbit ISL1 (ab109516, Abcam), rabitt-anti-Ki67 (ab15580, Abcam), rabbit-anti-GABA (A2052, Sigma Aldrich), mouse-anti-SMI32 (801701, BioLegend), rabbit-anti-MAP2 (AB5622, EMD Millipore), rabbit-anti-TUJ-1 (MRB-435P, BioLegend) Secondary antibodies were donkey and goat AlexaFluor488, 546, 555, and 650-conjugated antibodies raised against the appropriate species (used at 1:1000, Invitrogen). Where stated, the lipophilic membrane dye (FM 4-64FX, F34653, Molecular Probes) was used following the manufacturer’s instructions. Immunocytochemical images were acquired with an automated microscope (PerkinElmer Operetta), quantitative image analysis was performed using Columbus software (PerkinElmer) and Harmony High-Content Imaging software (PerkinElmer). Confocal images were acquired with a LSM 700 Inverted Confocal Microscope.
6
Immunofluorescence Microscopy for Protein Quantification
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Immunofluorescence staining was performed as described previously [37 (link)]. The images were examined with a laser-scanning confocal microscope (LSM 700, Carl Zeiss; Oberkochen, Germany). Quantitative analysis was performed by determining the immunofluorescence intensity of the target protein(s) using ImageJ [38 (link)] and normalized with the cell number on each slide. At least 100 cells were scored in each condition. The sources of the antibodies used in immunofluorescence staining are listed below: anti-γH2AX (Millipore; 05–636), anti-TUJ1 (Biolegend; MRB435P), and anti-TRAX [25 (link)].
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