Membranes were transferred into 6 well plates and slices were fixed for 20mins in 4 % paraformaldehyde in 0.1 M PBS (applied both above and below the membrane insert). To reduce the volumes required for subsequent steps, the membranes were cut free of the plastic inserts and the sections of membrane containing the slices were transferred, using forceps, to individual wells in a 24 well plate. Slices were washed twice in TBS, blocked for 1 h in blocking solution (TBS with 0.5 % Triton X-100 and 3 % Goat Serum) then incubated in 200 μl primary antibody diluted in blocking solution overnight at 4 °C with shaking. Slices were washed 3 times in TBS before being incubated (2 hs, RT in the dark) with Alexa488, 568 or 647 conjugated secondary antibodies (Life Technologies-diluted 1:250 in blocking solution). After a final 3 TBS washes, some slices were counterstained with Thioflavin S, BTA-1, Nissl or Hoechst. Images were captured using a Nikon Confocal Microscope. Primary antibodies used: mouse Tuj1 (Covance 1:1000), rabbit Tuj1 (Sigma 1:500), chicken Tuj1 (Abcam:1:1000) rabbit NFL (Millipore 1:250), mouse MOAB2 (pan specific to Aβ- Millipore 1:1000), rabbit GFAP (Abcam 1:1000), mouse synaptophysin (Dako 1:1000), rabbit PSD95 (Abcam 1:500), rabbit tau (Dako 1:1000), rabbit Iba1 (Wako 1:500) rabbit calbindin and rabbit parvalbumin (Kind gifts from Dr P Emson 1:1000).
Mouse tuj1
Manufactured by Fortrea
Sourced in United States
The Mouse Tuj1 is a laboratory equipment used for the detection and analysis of the Tuj1 protein, which is a marker for neuronal cells. The product provides a reliable and precise tool for researchers studying neuronal development and function.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit gfap, by Agilent Technologies (2 mentions)
Rabbit gfap, by Abcam (1 mentions)
Rabbit iba1, by Fujifilm (1 mentions)
Rabbit tuj1, by Merck Group (1 mentions)
Rabbit psd95, by Abcam (1 mentions)
Confocal microscope, by Nikon (1 mentions)
Rabbit psmad1 5 8, by Cell Signaling Technology (1 mentions)
Mouse gad67, by Merck Group (1 mentions)
Dapi fluoromount g, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Dojindo Laboratories (1 mentions)
Rabbit anti gfp, by Thermo Fisher Scientific (1 mentions)
Rabbit tuj1, by Fortrea (1 mentions)
Bx51 fluorescence microscope, by Olympus (1 mentions)
Fluorescence mounting medium, by Agilent Technologies (1 mentions)
Mouse anti ki67, by BD (1 mentions)
Mouse oct3 4, by Santa Cruz Biotechnology (1 mentions)
5 protocols using mouse tuj1
1
Immunohistochemical Analysis of Brain Slices
2
Immunohistochemistry of Neural Markers
3
Immunocytochemistry of Cortical Progenitors
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Cortical progenitors were cultured for 2 days to assess proliferation and for 3 days to analyze in vitro differentiation. The cells were fixed for 10 min in 4% PFA in 0.1 m phosphate buffer, permeabilized with 0.2% NP-40 for 10 min and blocked in 6% goat serum and 0.5% bovine serum albumin in phosphate-buffered saline (−) for 1 h. The slides were incubated with primary antibodies overnight at 4 °C, followed by 1-h incubation with secondary antibodies at room temperature. Mouse anti-Ki-67 (1:200, BD Bioscience Franklin Lakes, NJ, USA), chick anti-green fluorescent protein (GFP) (1:400, Thermo Fisher Scientific), rabbit anti-GFP (1:400, Thermo Fisher Scientific), mouse Tuj1 (1:1000, Covance, Princeton, NJ, USA) and rabbit Tuj1 (1:1000, Covance) were used as primary antibodies. Alexa 488 goat anti-rabbit and anti-mouse immunoglobulin G (1:500, Thermo Fisher Scientific), and Alexa 568 anti-rabbit, and anti-mouse IgG immunoglobulin G (1:500, Thermo Fisher Scientific) were used as secondary antibodies. Finally, we counterstained with 4',6-diamidino-2-phenylindole (DAPI; (1:1000, Dojindo, Kumamoto, Japan) and mounted the slides using Fluorescence Mounting Medium (Agilent Technologies, Santa Clara, CA, USA). Images were acquired using a BX51 fluorescence microscope (Olympus, Tokyo, Japan).
4
Protein Expression Profiling in Brain Cells
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OBs were microdissected from the brain, homogenized, and lysed in RIPA buffer (with Halt, 5 mM EDTA, and DNaseI) on ice. Cell pellets were thawed and lysed in Nonidet P-40 buffer (1% Nonidet P-40 buffer, 50 mM Tris HCl, pH 8.0, 150 mM NaCl) on ice for 45 min. Lysed cells were centrifuged for 20 min at 14,000 rpm in 4°C. The supernate was saved and concentration assessed at 595 nm using a spectrophotometer and the Protein Concentration Assay Reagent (Bio-Rad, Hercules, CA, USA) to generate standards. Twenty micrograms of total protein was loaded into a 10% SDS-PAGE gel and separated for 1 h at 200 V. A polyvinylidene fluoride membrane was activated, and proteins were transferred at 30 V for 1 h at 4°C. Membranes were blocked in 10% milk in 1× PBS for 1 h at room temperature. Primary antibodies were incubated overnight at 4°C and diluted in 5% milk in 1× PBS with Tween 20: mouse GFAP (1:1000; MilliporeSigma), mouse TuJ1 (1:1000; Covance, Princeton, NJ, USA), mouse Oct3/4 (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA), rat LIN28 (1:500), and mouse β-actin (1:1000; MilliporeSigma). Bound primary antibodies detected using horseradish peroxidase-conjugated anti-mouse (1:10,000) and anti-rat (1:12,500) were incubated for 1 h at room temperature and diluted in 5% milk in 1× PBS Tween 20.
5
Immunohistochemical Analysis of Neuronal Markers
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