The organoids were fixed and processed for immunostaining as is or after cryosectioning. Primary antibodies used for immunostaining were as follows: rabbit anti-SOX2 (Millipore), mouse anti-ISL1 (DSHB), rabbit anti-OLIG2 (IBL), mouse anti-NeuN (Chemicon), goat anti-NKX6.1 (R&D Systems), guinea pig anti-CHX1017 (link), rabbit anti-TUJ1 (BioLegend), mouse anti-TUJ1 (Millipore), rabbit cleaved-caspase3 (Cell Signaling Technology), goat anti-T (R&D Systems), rabbit anti-GFP (Abcam), goat anti-CHAT (Chemicon), and mouse anti-SMI-32 (BioLegend). After cell washes, samples were incubated with secondary antibodies and counterstained with Alexa Fluor® 488 phalloidin (Invitrogen) for axon labeling and Hoechst 33342 (Thermo Fisher Scientific) for nuclei staining.
Rabbit anti tuj1
Manufactured by BioLegend
Sourced in United States
Rabbit anti-Tuj1 is a primary antibody that binds to the Tuj1 (Neuron-specific Class III Beta Tubulin) protein. Tuj1 is a widely used marker for neurons and neuronal precursor cells. This antibody can be used to identify and visualize neurons in various applications.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti neun, by Merck Group (2 mentions)
Rabbit anti sox2, by Merck Group (1 mentions)
Mouse anti tuj1, by Merck Group (1 mentions)
Goat anti nkx6, by R&D Systems (1 mentions)
Mouse anti smi32, by BioLegend (1 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Rabbit cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Goat anti chat, by Merck Group (1 mentions)
Rabbit anti gfp, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Santa Cruz Biotechnology (1 mentions)
Rabbit anti map2, by Merck Group (1 mentions)
Mouse anti vgat, by Synaptic Systems (1 mentions)
Mouse anti pax6, by Merck Group (1 mentions)
Lsm 700, by Zeiss (1 mentions)
Mouse anti gfap, by Merck Group (1 mentions)
Rabbit anti olig2, by Merck Group (1 mentions)
Dm5000b, by Leica (1 mentions)
Mouse anti nestin, by Merck Group (1 mentions)
Mouse anti map2, by Merck Group (1 mentions)
6 protocols using rabbit anti tuj1
1
Immunostaining of Organoid Cultures
2
Comprehensive Immunofluorescence Profiling of Neural Cell Markers
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Cells were fixed with 4% paraformaldehyde (PFA) in PBS, permeabilized and blocked with 0.1% Triton-X100 plus and 5% normal goat serum in PBS. Then, they were incubated overnight at 4°C with the following primary antibodies: mouse anti-DLX2, 1:500 (Santa Cruz), rabbit anti-GABA, 1:1000 (Sigma), rabbit anti-GAD65/67, 1:250 (Millipore), mouse anti-GFAP, 1:200 (Millipore), mouse anti-MAP2, 1:200 (Chemicon), rabbit anti-MAP2, 1:1000 (Millipore), mouse anti-nestin, 1:200 (Chemicon), mouse anti-NeuN, 1:500 (Millipore), rabbit anti-neurofilament M, 1:200 (Millipore), mouse anti-NKX2.1, 1:1000 (Millipore), rabbit anti-OLIG2, 1:1000 (a gift from Dr. Charles Stiles, Harvard Medical School), mouse anti-PAX6, 1:250 (Millipore), mouse anti-PSD95, 1:500 (Millipore), rabbit anti-S100, 1:250 (Dako), mouse anti-SOX2, 1:500 (Millipore), rabbit anti-synaptophysin, 1:250 (Sigma), rabbit anti-TuJ1, 1:1000 (BioLegend) and mouse anti-vGAT, 1:200 (Synaptic Systems). After washing with PBS, cells were incubated with the secondary antibodies, Alexa Fluor® 555 anti-mouse IgG (Molecular Probes) and Alexa Fluor® 488 anti-mouse IgG (Molecular Probes). Cells were then counter-stained with 4,6-diamidino-2-phenylindole (DAPI) (Santa Cruz). The images were captured using a confocal laser-scanning microscope (LSM700; Zeiss) and digital inverted fluorescence microscope (DM5000B; Leica).
3
Immunofluorescence and Immunohistochemistry of Teratoma
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For IF, 5 μm of paraffin-embedded teratoma sections were pretreated with a proteinase K solution prior to incubation with rabbit anti-GFAP (1:1000; Dako). After washing, primary antibody was detected using donkey anti-rabbit-Cy3 (1:250; Jackson ImmunoResearch), washed, stained with Hoechst, and mounted in VectaShield (Vector Laboratories). The images were acquired on the Zeiss LSM 710 confocal microscope. For IHC, 5 μm of paraffin-embedded teratoma sections were pretreated with proteinase K, stained with rabbit anti-Tuj1 1:200 (BioLegend), washed, and detected using a donkey anti-rabbit-HRP. DAB (3,3′diaminobenzidine) was used for visualization. Nuclei were counterstained with hematoxylin (blue).
4
Immunostaining of Neuronal Cell Markers
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Cells were fixed with 4% formaldehyde for 10 minutes at room temperature, washed with PBS containing 0.1% Triton X-100 (PBST), blocked with 5% normal serum (Jackson ImmunoResearch, West Grove, PA, USA) in PBST for 30 minutes, incubated with primary antibody for 2 hours at room temperature, washed with PBST three times, incubated with secondary antibody for 1 hour at room temperature, washed with PBST three times, incubated with 4′6-diamidino-2-phenylindole to stain the nuclei for 5 minutes at room temperature, and washed with PBST three times. The stained cells were observed and imaged under a ZEISS (Carl Zeiss, Jena, Germany) Axio Observer Z1inverted microscope. The antibodies used were rabbit anti-TUJ1 (802001; BioLegend, San Diego, CA, USA), mouse anti-BRN3A (MAB1585; Millipore, Merck Millipore, Billerica, MA, USA), rabbit anti-NGN1 (ab66498; Abcam), chicken anti-GFP (ab13970; Abcam), rabbit anti-SYNAPSIN I (AB1543; Millipore), rabbit anti-vGLUT1 (135302; Synaptic System, Göttingen, Germany), rabbit anti-RECOVERIN (AB5585; Millipore), sheep anti-CHX10 (AB9014; Millipore), mouse HPC-1 (S0664; Sigma-Aldrich), rabbit anti-RBPMS (1830; PhosphoSolutions, Aurora, CO, USA), rabbit anti-NF200 (N4142; Sigma-Aldrich), and Alexa 488– or Alexa 568–conjugated secondary antibodies (Thermo Fisher).
5
Immunostaining of Scaffold Cultures
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Scaffolds were fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline solution (PBS) at room temperature (RT) for 15 min and then washed and stored in PBS at 4 °C. The following immunostaining protocol was used. Scaffolds were incubated for 1 h at room temperature with 10% donkey serum, 0.25% Triton X-100 in PBS. Primary antibodies were incubated in the same solution overnight at 4 °C, and secondary antibodies were incubated for 2 h at room temperature in 1% donkey serum, 0.25% Triton X-100 in PBS solution. Scaffolds were counterstained with Hoechst (H3570, Life technologies) for nuclei detection. Primary antibodies were used at the following dilutions: rabbit anti-Tuj1 (BioLegend, 1:1000) and goat anti-mCherry (Sicgen, 1:500). Secondary antibodies were from Jackson Immuno Research. Scaffolds were mounted in 1× PBS under a coverslip for analysis, and optical sections were obtained on a confocal microscope (SP5, Leica). The ImageJ software was used to generate Z-stacks and assemble pictures.
6
Culturing and Immunostaining Embryonic Cortical Neurons
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Cortices were microdissected from E18.5 embryos, digested in trypsin (Pierce), manually triturated and plated on 35mm well onto poly-D-lysine coated coverslips at a density of 150,000 cells for the neurite outgrowth assay or 500,000 for cellular localization experiments. Cells were plated in medium containing DMEM with 10% FBS and 1% penicillin/streptomycin and 4 hours after plating, media was completely removed and replaced with Neurobasal media containing B-27+ (Gibco/Invitrogen), 1% penicillin/streptomycin, and L-glutamine. Cells were cultured for 24 hours or 1 week in a 37°C incubator containing 5% CO2, then fixed for 10 minutes in 4% paraformaldehyde. Immunocytochemistry was conducted using the following antibodies: mouse anti-Tau (1:200; Abcam), rabbit anti-MAP2 (1:1000; Abcam), rabbit anti-Mllt11 (1:300, Abcam), rabbit anti-Tuj1 (1:1000, Biolegend), and mouse anti-acetylated α-tubulin (1:1000, Millipore Sigma).
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