Cell cultures were fixed with 4% paraformaldehyde (PFA, 15 min) after the completion of the respective experiment. After fixation, immunocytochemistry was conducted by first washing the cells with DPBS 3 times, then incubating in 5% normal horse serum for 60 min to block nonspecific binding (VWR, 102643-676). Afterwards, the cells were incubated in 1:500 TUBB3 (BioLegend, 801202) for 16 h. followed by another 3 washes with DPBS and incubation in 1:500 Alexa Flour 488 (ThermoFisher Scientific, A32723) and 1:10,000 DAPI (VWR, 95059-474) for 60 min, all conducted at room temperature. After the completion of immunocytochemistry, the cells were preserved in Fluoromount-G Mounting Medium (ThermoFisher Scientific, 00-4958-02) until imaging. All experimentation after incubation in secondary antibody was performed in the absence of light.
Tubb3
Manufactured by BioLegend
Sourced in United States
TUBB3 is a protein that functions as a component of microtubules, which are cytoskeletal structures involved in various cellular processes. It is commonly used as a marker for neuronal cells and their differentiation.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
β actin, by Merck Group (2 mentions)
Fluoromount g mounting medium, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Avantor (1 mentions)
Alexa flour 488, by Thermo Fisher Scientific (1 mentions)
Ctip2, by Abcam (1 mentions)
Cleaved caspase 3 asp175 antibody, by Cell Signaling Technology (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 secondary, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Glial fibrillary acidic protein (gfap), by Agilent Technologies (1 mentions)
Prolong gold antifade mounting media, by Thermo Fisher Scientific (1 mentions)
Metamorph software, by Molecular Devices (1 mentions)
Alexa fluor 488 goat anti mouse igg h l a11001, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 goat anti rabbit igg h l a11012, by Thermo Fisher Scientific (1 mentions)
Normal donkey serum (nds), by Jackson ImmunoResearch (1 mentions)
Ab79415, by Abcam (1 mentions)
Anti stat1, by Cell Signaling Technology (1 mentions)
P0217, by Agilent Technologies (1 mentions)
Anti p stat1, by Cell Signaling Technology (1 mentions)
14 protocols using tubb3
1
Immunocytochemical Neuronal Staining Protocol
2
Immunolabeling of Neural Markers in Cell Cultures
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Primary antibodies were Pax6 (Mouse, BD Pharmigen, cat#561462, dilution 1:100), Pax6 (Rabbit, Abcam, cat#ab195045, dilution 1:300), Sox2 (Rabbit, Merck, cat#AB5603, dilution 1:200), TBR1 (Rabbit, Abcam, cat#ab31940, dilution 1:100), TBR2 (Rabbit, Abcam, cat#ab23345, dilution 1:100), TUBB3 (Mouse, BioLegend, cat#801201, dilution 1:300), MAP 2 (Mouse, Merck, cat#MAB3418, dilution 1:50), CTIP2 (Rat, Abcam, cat#ab18465, dilution 1:100) and Cleaved Caspase-3 (Asp175) Antibody (Cat# 9661, Cell Signaling Technology, dilution 1:100). Secondary antibodies were species-specific antibodies conjugated with AlexaFluor 488, 546, 594 or 647 (Invitrogen).
3
Immunostaining of Cochlear Hair Cells
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Antibodies, dilutions and conditions used for immunostaining of iMOP cells were previously described (Kwan et al., 2015 (link)). MYO6 (Proteus Biosciences Inc.), GFAP (Dako) and TUBB3 (BioLegend) antibodies were purchased from commercial sources. Cochlear explants were fixed in 4% formaldehyde with 1× PBS for 1 h, permeabilized in wash buffer (PBS and 0.1% Triton X-100) for 10 min, incubated in blocking buffer (PBS, 10% goat serum and 0.1% Triton X-100) for 1 h and incubated overnight with a 1:500 dilution of MYO7A (Proteus Biosciences Inc.) primary antibody in blocking buffer. Cochleae were rinsed in wash buffer before incubating with a 1:500 dilution of goat anti-rabbit Alexa Fluor 488 secondary antibody and 1:500 phalloidin Alexa Fluor 647 (Life Technologies) in blocking buffer for 1 h. Cochleae were washed and mounted on slides with prolong gold antifade mounting media (Life Technologies). Immunofluorescence images were obtained using either a Zeiss 510 confocal microscope with a 40×1.3 NA water immersion objective or an Olympus DSU unit with a 60×1.3 NA apochromatic oil immersion objective. Conventional fluorescent filter sets were used. The relative percent of MYO7A positive inner hair cells (IHC) and outer hair cells (OHC) were obtained by calculating the ratio of MYO7A cells counted along the cochlear axis from experimental and control animals.
4
Immunochemical Analysis of Neuronal Phenotype
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Neurons were cultured on a coverslip and washed with PBS. Subsequently, they were fixed with 4% paraformaldehyde for 20 min at room temperature. After the residual fixation buffer was removed using PBS, the cells were blocked in PBS containing 5% BSA for 20 min at room temperature and then hybridized with the primary antibody tubulin class III (TUBB3) (1 : 5000 dilution, BioLegend) overnight at 4°C. After being washed with PBS, the cells were incubated with an Alexa 488-conjugated secondary antibody in the dark for 1 hour. For cell counting, the cells were stained with 4′,6-diamidino-2-phenylindole (1 : 1000 dilution). Subsequently, the mounted coverslips were examined using a Leica TCS confocal microscope. Neurite outgrowth on the SH-SY5Y-derived neurons (>200 cells) was analyzed using MetaMorph software (Molecular Devices).
5
Immunofluorescence Staining of Cytokine Receptors
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After 24 h of cytokine/inflammatory mediator treatment, cells were fixated (4% in paraformaldehyde (PFA) in phosphate-buffered saline (PBS)) and permeabilized (0.2% Titron X-100 in PBS; ThermoFisher Scientific) for immunofluorescence staining. Monoclonal antibodies against Class III ß-tubulin (TUBB3; 657,402, BioLegend), microtubule-associated protein 2 (MAP2; sc-74421, Santa Cruz), IL-17RA (clone G9, sc-376374, Santa Cruz), IL-10RB (clone F6, sc-271969, Santa Cruz), TNFR1 (clone H-5, sc-8436 Santa Cruz) or IFNGR1 (clone GIR 94, sc-12755, Santa Cruz) were added, and cells were incubated for 1 h at room temperature. Afterwards, matching secondary antibodies (Alexa Fluor™ 594 goat anti-mouse IgG(H + L) A11032, Alexa Fluor™ 488 goat anti-mouse IgG(H + L) A11001, Alexa Fluor™ 488 donkey anti-rabbit IgG(H + L) A21206, Alexa Fluor™ 594 goat anti-rabbit IgG(H + L) A11012, all from Invitrogen) each in a dilution of 1:1000 were applied respectively for 1 h at room temperature and DAPI (4’,6-diamidino-2-phenylindole) was used for nuclear staining. Primary antibodies were used at the following concentrations in 1% BSA/PBS upon application: mouse anti hTUBB3 (1:250), rabbit anti hMAP-2 (1:100), anti hIL-17RA (1:100), anti hIL-10RB (1:100), anti hTNFR1 (1:100), anti hIFNGR1 (1:100).
6
Immunohistochemistry Analysis of Myelin and Axons
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After dRNA-HybISS RNA detection, tissue was blocked with PBTA (PBS, 5% normal donkey serum (Jackson ImmunoResearch), 0.5% Triton-X 100) for one hour. Then sections were incubated with primary antibodies, either MBP (Abcam, ab7349) or TUBB3 (BioLegend, 801,213) overnight at + 4 °C. Sections were then washed three times with PBS and incubated with secondary antibodies (Alexa Fluor anti-rat 488 and anti-mouse 555) for 2 h at room temperature and counterstained with DAPI.
7
Comprehensive Immunoblotting Protocol
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Anti‐α‐tubulin (T9026 Sigma‐Aldrich Co.), anti‐neuron‐specific class III β‐tubulin (Tuj1, TUBB3 #80120, BioLegend) anti‐acetylated tubulin (T7451 Sigma‐Aldrich), anti‐GAPDH (MAB374 Merck Millipore) anti‐PKR (anti‐EIF2AK2 Antibody No. ABIN500507, antibodies‐online.com), anti‐V5 tag antibody (SV5‐Pk1, Abcam), anti‐tau antibody (in house produced polyclonal Tau antibody, affinity‐purified rabbit towards human 2N4R tau), anti‐tau (A0024, DAKO), anti‐STAT1 (#9172 Cell Signaling Technology), anti‐pSTAT1 (pS727, #9177 Cell Signaling Technology), anti‐pS199/202 tau (44‐768G Thermo Fisher Scientific), anti‐pS396 tau (44‐752G Thermo Fisher Scientific), AT8 (pS202/T205 tau, MN1020 Thermo Fisher Scientific), AT180 (pT231 tau, MN1040 Thermo Fisher Scientific), AT270 (pT181 tau, MN1050 Thermo Fisher Scientific), anti‐pS262 (44‐750G Thermo Fisher Scientific), anti‐pS404 (44‐758G Thermo Fisher Scientific), anti‐pS409 (Lu0041G, kindly provided by Lundbeck) and anti‐pS422 (ab79415, Abcam) were used in accordance with the manufacturers' recommendations. HRP‐conjugated rabbit immunoglobulins (#P0217, Dako) and secondary HRP conjugated mouse immunoglobulins (#P0260, Dako) were used as secondary antibodies.
8
Western Blotting Analysis of Cellular Proteins
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For Western blotting analysis, cells were lysed as described previously [43 (link)]. Primary antibody, TUBB3 (BioLegend, San Diego, CA, USA), PTEN, Akt, phosphorylation Akt (p-Akt), and cleaved PARP (Cell Signaling Technology, Inc., Danvers, MA, USA) were used. β-Actin (Sigma-Aldrich, St. Louis, MO, USA) was used as a loading control. The ID and dilution of primary and secondary antibodies are summarized in Table 1 .
9
Assessing Neurite Outgrowth in ΔK280 Tau-Expressing SH-SY5Y Cells
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As described, ∆K280 tauRD-DsRed SH-SY5Y cells were seeded in a 24-well plate (5 × 104/well) with retinoic acid addition on day 1, treated with tested compounds (10 µM) and ∆K280 tauRD-DsRed expression was induced with doxycycline (2 μg/mL) on day 2. On day 8, after being fixed in 4% paraformaldehyde for 15 min, permeabilized in 0.1% Triton X-100 for 10 min and blocked in 3% bovine serum albumin (BSA) for 20 min, the cells were stained with TUBB3 (neuronal class III β-tubulin) primary antibody (1:1000; BioLegend, San Diego, CA, USA) at 4 °C overnight, followed by goat anti-rabbit Alexa Fluor® 555 secondary antibody (1:1000; Molecular probes) at room temperature for 2 h, with 4′-6-diamidino-2-phenylindole (DAPI, 0.1 µg/mL; Sigma-Aldrich) included for nuclei staining. Neuronal images were captured using the high-content analysis system as described. Neurite total length (μm), processes (primary neurite extensions projecting directly from the cell body) and branching (points at which primary neurites bifurcated) were analyzed using Neurite Outgrowth Application Module (MetaXpress; Molecular Devices). Around 5000 cells were analyzed in each of three independent experiments for each sample.
10
Immunohistochemical Analysis of Neuronal Cultures
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CxV explants were fixed at 4–6 div for 10 min in 4% PFA (Sigma), washed with PBS and permeabilized for 10 min with 0.2% Triton-X in PBS. The following primary antibodies were used: Gad67 (Chemicon), GAP-43 (Abcam), GFAP (Sigma), mO4 (gift from J. Trotter), PKCγ (Santa Cruz Biotechnology), phospho-GAP-43 (Thermo Fischer), phospho-PKCγ (Biozol), PTEN Cascade Biosciences), PV (Swant), SMI-32 (Covance), and β-III-Tubulin (Tubb3, mouse, Covance; rabbit, Abcam). For human neuronal cultures IL-4R (BD Pharmingen), Homer-1/2/3 (Synaptic Systems), IRS1 (Abbexa), phospho-IRS1 (pIRS1, Abbexa), NeuN (Merck Millipore), PKCγ (Abcam), Neurofilament light chain (Abcam), MAP2 (Abcam), Synaptophysin (Synaptic Systems), and Tubb3 (BioLegend), were used in addition. After washing with PBS, incubation was performed for 1 h with Alexa-conjugated anti-mouse and anti-rabbit secondary antibodies (Life Technologies), Alexa 488-conjugated Phalloidin (Thermo Fisher) and dapi (Invitrogen).
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