Cells from neurosphere differentiation cultures were fixed in 4% PFA with a 30% sucrose solution for 30 min at 37°C. For immunocytochemistry, cultures were preincubated for 1 h in blocking solution (10% goat serum, 0.1% Triton X-100, BSA), followed by overnight incubation with the appropriate primary antibody at 4°C. The following primary antibodies were used: mouse anti-hGFAP (1:500, Sternberger Monoclonal), chicken anti-vimentin (1:200, Millipore), rabbit anti-NG2 (1:200, Millipore), mouse anti-Olig2 (1:200, Millipore), chicken anti-Tuj1 (1:200, Millipore), rabbit anti-active caspase-3 (1:200, Abcam), rabbit anti-HSP27 (1:200, Abcam), and rabbit anti-cathepsin (1:200, Abcam). The corresponding secondary antibodies were incubated for 2 h (Alexa-Fluor 405, 488, 555, or 647 goat anti-mouse, chicken or rabbit; 1:500; Invitrogen), followed by incubation with DAPI (1:1,000, Sigma) for 10 min and rinsing before being mounted on glass slides with Fluorsave (Calbiochem). Analyses were performed with a Nikon 80i fluorescence microscope at 40× or 63× magnification.
Chicken anti tuj1
Manufactured by Merck Group
Chicken anti-TUJ1 is a laboratory antibody product. It is used to detect the presence of the TUJ1 protein, which is a marker for neuronal cells. The antibody is derived from chicken and is designed for use in various research and diagnostic applications.
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Lab products found in correlation
Axio observer z1, by Zeiss (3 mentions)
Lsm 710, by Zeiss (2 mentions)
Chicken anti vimentin, by Merck Group (1 mentions)
80i fluorescence microscope, by Nikon (1 mentions)
Rabbit anti ng2, by Merck Group (1 mentions)
Rabbit anti active caspase 3, by Abcam (1 mentions)
Mouse anti olig2, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Mouse anti tuj1, by Promega (1 mentions)
Rabbit anti nurr1, by Santa Cruz Biotechnology (1 mentions)
Goat anti chl1, by R&D Systems (1 mentions)
Chicken anti th, by Abcam (1 mentions)
Rabbit anti th, by Abcam (1 mentions)
D9663, by Merck Group (1 mentions)
Fluoromount g mounting medium, by Southern Biotech (1 mentions)
Nuclear dye hoechst 33342, by Merck Group (1 mentions)
Vt1000, by Leica (1 mentions)
3 protocols using chicken anti tuj1
1
Immunocytochemical Analysis of Neural Cells
2
Immunohistochemistry of Developmental Embryos
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Embryos were fixed in 4% paraformaldehyde (2–8 hrs, dependent on developmental age), cryopreserved in 20% sucrose and coronally sectioned (16 μm, 1:10 series). For primary cultures and explant assays, cells were fixed with 4% paraformaldehyde, rinsed and stored in PBS with 0.03% sodium azide.
Immunohistochemistry was performed using previously described methods21 (link). Antibodies used were as follows: goat anti-CHL1 (1:500, R&D Systems), rabbit anti-NURR1 (1:200, Santa Cruz), rabbit anti-TH (1:800, PelFreez), chicken anti-TH (1:400, Abcam), mouse anti-TUJ1 (1:15000, Promega), and chicken anti-TUJ1 (1:200, Millipore). Appropriate secondary antibodies, donkey 488, 555 and 647 Alexaflour (Jackson ImmunoResearch Laboratories), were used at a dilution of 1:400. Images were captured using fluorescence microscope (Zeiss Axio Observer Z1 or Zeiss 780 confocal microscrope).
Immunohistochemistry was performed using previously described methods21 (link). Antibodies used were as follows: goat anti-CHL1 (1:500, R&D Systems), rabbit anti-NURR1 (1:200, Santa Cruz), rabbit anti-TH (1:800, PelFreez), chicken anti-TH (1:400, Abcam), mouse anti-TUJ1 (1:15000, Promega), and chicken anti-TUJ1 (1:200, Millipore). Appropriate secondary antibodies, donkey 488, 555 and 647 Alexaflour (Jackson ImmunoResearch Laboratories), were used at a dilution of 1:400. Images were captured using fluorescence microscope (Zeiss Axio Observer Z1 or Zeiss 780 confocal microscrope).
3
Immunostaining of Cultured hMOs
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Cultured and fixed hMOs were embedded in a 3% lowmelting-point agarose (Biozym) in PBS. Subsequently, 50 µm thick sections were cut using a vibratome (Leica VT1000s) and center-sections were used for assessing TH/FOXA2/TUJ1 expression. Prior to the immunostaining, sections were permeabilized using 0.5% Triton X-100 in PBS. Depending on the antibody, permeabilization times varied between 30 min and 2 h. Unspecific antigen blocking was achieved by incubating cut sections for 2 h in 2.5% donkey serum (Sigma-Aldrich, D9663), 2.5% BSA, 0.1% Triton X-100 and 0.1% sodium azide, followed by primary antibody incubation at 4 °C for 48 h on a shaker. Antibodies were diluted in blocking buffer as follows: rabbit anti-TH (1 : 1000, Abcam), chicken anti-TUJ1 (1 : 600, Millipore). This was followed by the incubation with secondary antibodies diluted in PBS containing 0.01% Triton X-100 and Hoechst-33342 nuclear dye (1 : 1000, Sigma-Aldrich). All secondary antibodies (Invitrogen) were conjugated to Alexa Fluor fluorochromes. Sections were mounted in Fluoromount-G mounting medium (Southern Biotech) and analyzed employing a confocal laser scanning microscope (Zeiss LSM 710).
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