Anti-beta III Tubulin antibody [TU-20] - Neuronal Marker (ab7751)

Naive hOMSC express constitutively neuronal and dopaminergic markers.

Immunofluorescence of neuronal (A–C) and dopaminergic (D–F) markers in naïve hOMSC, scale bars 100 µM.

Cells obtained from two donors (24 and 35 years old) were fixed in 4% PFA-PBS and pre-incubated for 60 min in blocking solution (5% goat serum, 1% BSA, 0.05% Triton-X in PBS). Primary antibodies were diluted in the blocking solution and applied overnight at 4°C. The following primary antibodies were used, β-III-Tubulin (1∶200, ab7751, abcam), Map2 (1∶200), synapsin (1∶200), tyrosine hydroxylase (1∶200), Lmx1A (1∶200), Pitx3 (1∶200) and Nurr1 (1∶200).

Primary antibodies were detected with fluorescent-labeled secondary antibodies Alexa^®488 and 568 (1∶500,) for 1 h at room temperature. Nuclear DNA was stained using 4,6-diamino-2-phenylindole (DAPI) (1∶1000).

Kif2a is involved in the differentiation of NSCs/NPCs.

(Panel A) Mouse embryos were electroporated with indicated plasmids (scramble shRNA, Kif2a shRNA1 or Kif2a shRNA1+Kif2a^Res) at E13.5, and analyzed at E15.5. GFP (green) represents cells expressing the indicated plasmids; TU20 (ab7751, red) represents immature neurons. Scale bars = 50 μm.

Confirmation of differentiation of iPSCs into cortical neuronal cultures.

Images taken from clones C2 (trisomic) and C3 -D21 (disomic) 40 days after initiation of the differentiation protocol. Fixed cells on chamber slides were probed with antibodies against the marker proteins listed in the left column. Neuronal marker Beta III tubulin is red (ab7751); DAPI is blue; and Ctip2, TBR1, SLC17A7 (vGLUT1) and GRIK2 are green. Size bar = 20 μ.

ab7751 staining beta III Tubulin in Neuro-2a (Mouse neuroblastoma cell line) cells.

The cells were fixed with 100% methanol (5min) and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated with ab7751 at 1/1000 and ab6046 at 1µg/ml overnight at +4°C, followed by a further incubation at room temperature for 1h with an AlexaFluor^®488 Goat anti-Mouse secondary (ab150117) at 2 μg/ml (shown in green) and AlexaFluor^®594 Goat anti-Rabbit secondary (ab150088) at 2 μg/ml (shown in pseudo color red). Nuclear DNA was labeled in blue with DAPI.
Negative controls: 1– Rabbit primary and anti-mouse secondary antibody; 2 – Mouse primary antibody and anti-rabbit secondary antibody. Controls 1 and 2 indicate that there is no unspecific reaction between primary and secondary antibodies used.

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using LI-COR^® blocking buffer before being incubated with ab7751 overnight at 4°C. Antibody binding was detected using the IRDye^® 800CW Goat Anti-Mouse secondary at a 1:10,000 dilution for 1hr at room temperature and then imaged using the Odyssey^® CLx Imaging System.

IHC image of ab7751 staining beta III Tubulin in human cerebellum formalin fixed paraffin embedded tissue sections, performed on a Leica Bond.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab7751, 5μg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with hematoxylin and mounted with DPX. No primary antibody was used in the secondary only control (shown on the inset).
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

ab7751 staining beta III Tubulin in induced neurons derived from human pluripotent cells by ICC/IF (Immunocytochemistry/immunofluorescence).

Cells were fixed with paraformaldehyde, permeabilized with 0.25% Triton X-100 in PBS and blocked with 1% BSA for 30 minutes at 22°C. Samples were incubated with primary antibody (1/500) for 16 hours at 4°C. An Alexa Fluor^® 594-conjugated Donkey anti-mouse IgG polyclonal (1/1000) was used as the secondary antibody.

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Overlay histogram showing SH-SY5Y (Human neuroblastoma cell line from bone marrow) cells stained with ab7751 (red line).

The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab7751, 1/50 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight^® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

This antibody gave a positive signal in SH-SY5Y cells fixed with 80% methanol (5 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.

ab7751 staining neuron specific beta III Tubulin in human colon tissue sections by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections).

Tissue underwent formaldehyde fixation before heat mediated antigen retrieval in citric acid pH 6 and then blocking with 1% BSA was performed for 10 minutes at RT. The primary antibody was used at dilution at 1/200 and incubated with sample in TBS/BSA/azide for 2 hours. A biotin conjugated goat polyclonal to mouse IgG was used at dilution at 1/200 as secondary antibody. Images A and B represent staining of the clone TU20 and 2G10 respectively in ganglia of Auerbach's plexus.

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Immunohistochemistical detection of neuron specific beta III Tubulin using antibody (ab7751) on whole mouse e12 embryo (formaldehyde fixed/frozen sections).

Permeabilization: No. Blocking step: 1% BSA for 10 mins @ rt°C. Primary Antibody Dilution 1/200; Incubation time 2 hours in TBS/BSA/azide. Secondary antibody: anti Mouse IgG Conjugated to Alexa Fluor^® 594 (1/1000).

Floorplate region of developing cervical spinal cord (I have not studied this particular region).

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ab7751 immunostaining (1/500) neuronal processes in primary mouse cortical culture.

Immunohistochemical detection with neuron specific beta III Tubulin antibody [TU-20] (ab7751) on formaldehyde fixed rat spinal cord tissue sections.

Antigen retrieval step: heat mediated in citric acid pH 6. Blocking: 1% BSA for 10 mins@ rt°C. Primary antibody ab7751 incubated at 1/500 for 2 hours. Secondary antibody: anti mouse IgGs conjugated to biotin (1/200).

ab7751 shows good fibrillar positivity in rat spinal motor neurone cell bodies. Some L/S nerve fibre processes are clearly seen yet the majority in the white matter are also not obvious.

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