iPS cells were maintained and differentiated as previously described (see Supplementary data ) 37 (link). At day 18 neural precursor cells (NPCs) were isolated using Anti-PSA-NCAM MicroBeads (30-092-966¸ Miltenyi Biotec) according to the manufacturer’s protocol. Sorted cells were fluorescently stained with an Anti-IgM antibody conjugated with PE dye (130-095-908; Miltenyi Biotec) and analysed by flow cytometry using a GUAVA EasyCyte 6-2L instrument to assess purity of the recovered population. Unlabelled cells were used as negative control. Cultures were harvested at day 70 for analysis of mature neurons. To obtain a more pure population of neurons for qRT-PCR, mature cultures were sorted using a CD24 MicroBead Kit (130-095-951, Miltenyi Biotec)39 (link). Sorted neurons were plated on poly-ornithine and laminin coated slides and allowed to attach for 48 hours before being stained for β-III-Tubulin (Chemicon) to confirm their identity.
β tubulin 3
Manufactured by Merck Group
Sourced in United States, United Kingdom, Japan
β-tubulin III is a member of the tubulin protein family, which are the building blocks of microtubules. It is a key component of the cytoskeleton and plays a crucial role in cell structure and division. β-tubulin III is commonly used in laboratory settings for research related to cellular processes and neuronal development.
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Lab products found in correlation
Nestin, by Merck Group (6 mentions)
Glial fibrillary acidic protein (gfap), by Merck Group (5 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (4 mentions)
Glial fibrillary acidic protein (gfap), by Agilent Technologies (3 mentions)
Axio observer z1, by Zeiss (3 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (3 mentions)
Nanog, by R&D Systems (2 mentions)
Paraformaldehyde (pfa), by Merck Group (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
β actin, by Merck Group (2 mentions)
Alexa 647, by Thermo Fisher Scientific (2 mentions)
A 11030, by Thermo Fisher Scientific (2 mentions)
P40101, by Agilent Technologies (2 mentions)
Alexa 546, by Thermo Fisher Scientific (2 mentions)
Tcs sp2, by Leica (2 mentions)
Ab15592, by Abcam (2 mentions)
Ab109429, by Abcam (2 mentions)
Ab18189, by Abcam (2 mentions)
Ps1 ntf, by Merck Group (2 mentions)
Ps1 ctf, by Merck Group (2 mentions)
45 protocols using β tubulin 3
1
Efficient Isolation and Characterization of Mature Neurons
2
Immunocytochemistry for Neuronal Markers
3
Immunocytochemistry Analysis of Pluripotency and Differentiation Markers
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Cells were fixed with PBS containing 4% paraformaldehyde for 10 minutes at room temperature. After washing with PBS, the cells were treated with PBS containing 5% donkey serum, 1% bovine serum albumin (BSA, WAKO) and 0.1% Triton X‐100 for 45 minutes at room temperature. The cells were incubated with primary antibodies 4°C overnight and then stained with secondary antibodies. The primary antibodies included Sendai virus (MBL, Nagoya, Japan, PD029), OCT3/4 (BD, 611202), NANOG (R&D Systems, AF1997), β‐III‐tubulin (Chemicon, Billerica, MA, MAB1637), α‐SMA (DAKO, Santa Clara, CA, M0851) and SOX17 (R&D systems, AF1924). All the secondary antibodies (Alexa Fluor 594‐conjugated anti‐rabbit, ‐mouse, ‐goat IgG and Alexa Fluor 488‐conjugated anti‐mouse, ‐goat IgG were obtained from Life technologies. Hoechst 33342 (WAKO) was used for nuclear staining.
4
Immunocytochemical Analysis of Neuronal Markers
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On the 7th and 14th days after induction, cells on wells containing TCPS, CCh, CCh-Ca, and calcium were fixed with 4% paraformaldehyde (Sigma, USA) for 20 min. Human ADSCs were then permeabilized with 0.4% Triton X100 in PBS for 10 min 5% goat serum/PBS-tween-20 was used to incubate fixed cells for 30 min at 37 °C. Human ADSCs were then reacted overnight at 4 °C with the primary antibodies, including γ-enolase (1:100 Santa Cruz Biotechnology), MAP2 (1:300 Santa Cruz Biotechnology, INC), and β–III–tubulin (1:50 Chemicon) diluted in PBS/0.2% (v/v) bovine serum albumin. After incubation, the cells were rinsed three times with PBS tween-20 (0.1%). The hADSCs were incubated with the secondary antibody, phycoerythrin PE-conjugated anti-mouse IgG (1:100 Sigma), for 1 h at room temperature. DAPI (Sigma) was used to color the nuclei and images were prepared with a fluorescence microscope (Nikon, Japan). The quantitative assessment of the immunocytochemical intensity of γ-enolase, MAP2, and β–III–tubulin was done using image analysis software (ImageJ).
5
Immunostaining Protocol for Cell Characterization
6
Retinal Protein Localization through Immunostaining
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Formalin-fixed retinal explants were incubated overnight at −30°C in DMSO-ethanol (1:4 dilution) to promote penetration of antibodies. Retinal explants were subjected to two cycles of freezing and thawing between −80°C and room temperature in 100% ethanol for 20 minutes. Then retinas were rehydrated in 70%, 50%, and 15% ethanol and PBS for 20 minutes each and incubated overnight with 0.2% Triton X-100. Samples were stained with antibodies for SBDP150 as described above and β-III tubulin (as a neuron marker, 1:500 dilution; Merck). The NFL was observed at approximately 5 mm from the tips of the fan-shaped retinal flat mounts with a model SP5 AOBS spectral confocal system (Leica, Wentzler, Germany) or a model LSM 780 spectral confocal system (Carl Zeiss, Oberkochen, Germany).
7
Immunofluorescence and Apoptosis Analysis of Primary Neurons
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For immunofluorescence analysis, primary neurons seeded on coated coverslips were processed and confocal imaging was performed using a Nikon A1 confocal laser scanning microscope, as previously described [26 (link)].
Primary antibodies to detect βTubulin III (Millipore, CA, USA), pTauser422 (OriGene Technologies, MD, USA), and 6E10 (Bio Legend, CA, USA) were used following datasheet recommended dilutions. Alexa secondary antibodies (Thermo Fisher Scientific, Waltham, MA, USA) were used at 1 : 200 dilution.
The confocal serial sections were processed with ImageJ software to obtain three-dimensional projections. The image rendering was performed by Adobe Photoshop software.
For apoptosis detection, after 3 washes with 50 μl of binding buffer (10 mM HEPES, pH 7.5, containing 140 mM NaCl, and 2.5 mM CaCl2), the specimen was incubated for 15 minutes with 50 μl of double staining solution (binding buffer containing 0.25 μl of annexin V-FITC and 0.25 μl of propidium iodide (PI); BD Pharmingen™, Erembodegem, Belgium). Finally, the specimen was washed 5 times with 50 μl of binding buffer, mounted with 15 μl of binding buffer, and visualized under fluorescence microscopy [24 (link)].
Primary antibodies to detect βTubulin III (Millipore, CA, USA), pTauser422 (OriGene Technologies, MD, USA), and 6E10 (Bio Legend, CA, USA) were used following datasheet recommended dilutions. Alexa secondary antibodies (Thermo Fisher Scientific, Waltham, MA, USA) were used at 1 : 200 dilution.
The confocal serial sections were processed with ImageJ software to obtain three-dimensional projections. The image rendering was performed by Adobe Photoshop software.
For apoptosis detection, after 3 washes with 50 μl of binding buffer (10 mM HEPES, pH 7.5, containing 140 mM NaCl, and 2.5 mM CaCl2), the specimen was incubated for 15 minutes with 50 μl of double staining solution (binding buffer containing 0.25 μl of annexin V-FITC and 0.25 μl of propidium iodide (PI); BD Pharmingen™, Erembodegem, Belgium). Finally, the specimen was washed 5 times with 50 μl of binding buffer, mounted with 15 μl of binding buffer, and visualized under fluorescence microscopy [24 (link)].
8
In vivo Comparison of Odontogenic Capacities
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Cell sheets combined with human TDM were transplanted into the dorsum of immunodeficient mice (8-week-old males, n=9) under general anesthesia to further compare the odontogenic capacities of SHEDs and DFCs in vivo. Nine immunodeficient mice were randomly divided into three groups: (1) SHEDSs combined with TDM, (2) DFCSs combined with TDM, and (3) TDM alone. Each mouse was implanted with two samples. Eight weeks later, all samples were harvested from the immunodeficient mice, fixed with 4% paraformaldehyde overnight, demineralized with 10% EDTA (pH 7.6) at 37 °C for 3 months, and embedded in paraffin. Paraffin sections were prepared and subjected to hematoxylin and eosin (H&E), Masson's trichrome, and immunohistochemical staining.
Antibodies used for immunohistochemistry included DMP-1 (1:250 dilution, Millipore, USA), DSP (1:200 dilution, Santa Cruz, USA), COL-1 (1:200 dilution, Abcam, UK), OCN (1:500 dilution, Zenbio, China), Periostin (1:500 dilution, Santa Cruz, USA), TGF-β1 (1:200 dilution, Abcam, UK) and β-tubulin III (1:200 dilution, Millipore, USA). All antibodies were used according to the manufacturers' protocol. PBS was used in place of the primary antibodies as the negative control and secondary antibodies were visualized using the DAB kit (Gene Tech, China).
Antibodies used for immunohistochemistry included DMP-1 (1:250 dilution, Millipore, USA), DSP (1:200 dilution, Santa Cruz, USA), COL-1 (1:200 dilution, Abcam, UK), OCN (1:500 dilution, Zenbio, China), Periostin (1:500 dilution, Santa Cruz, USA), TGF-β1 (1:200 dilution, Abcam, UK) and β-tubulin III (1:200 dilution, Millipore, USA). All antibodies were used according to the manufacturers' protocol. PBS was used in place of the primary antibodies as the negative control and secondary antibodies were visualized using the DAB kit (Gene Tech, China).
9
Immunocytochemistry of Stem Cells
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For AP staining, cells were fixed with 90% alcohol for 2 min and washed three times with PBS and stained with BCIP/NBT for 30 min in the darkness. For immunostaining, cells were fixed in 4.0% paraformaldehyde for 20 min, permeabilized with 0.5% Tween-20 for 30 min, incubated with primary antibody overnight, and incubated with secondary antibody (Invitrogen) for 1 hour. The nuclei were counterstained by DAPI. The cells were imaged with an inverted confocal microscope. The primary antibodies used in this study were OCT4 (1 : 500, Abcam, Cambridge, MA, USA), nestin (1 : 100, Millipore), GFAP (1 : 500, Millipore), and β-tubulin III (1 : 500, Millipore).
10
Cranial Cartilage and Gene Expression Analysis in Xenopus Embryos
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The cranial cartilage staining was performed as previously described (Shi et al., 2014 (link)). For immunohistochemistry, embryos were collected at stage 40 and fixed with paraformaldehyde. The frozen samples were sectioned in 10 μm thickness, and stained with β-tubulin III (Sigma T8660). Total RNA samples were extracted with Trizol kit (Tiangen) and reversely transcribed with Fermentas RevertAid First Strand cDNA synthesis kit (Thermoscientific 1622). The RT-PCR primers for Fgf8, Wnt8, Dlx3, Dlx5, AP2α, Pax3, Msx1, Zic1, Zic5, Slug, Hes5.2a, Hes5.2b, ESR1, and Histone4 (H4) were listed in Table 1 .
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