Anti-beta III Tubulin antibody - Neuronal Marker (ab18207)

ab18207 staining beta III Tubulin in PC12 cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Tween for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab18207 at 1µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was acquired with a confocal microscope (Leica-Microsystems TCS SP8) and a single confocal section is shown.

ab18207 staining beta III Tubulin in primary rat neurons/glia, DIV14 (prepared from E18 rat hippocampal brain area, obtained from Transnetyx Tissue by BrainBits, LLC, cat.no. SDHEP) cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Tween for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab18207 at 1µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Also suitable in cells fixed with 4% paraformaldehyde (10 min).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

Lane 1: Wild-type HAP1 whole cell lysate (20 µg)
Lane 2: Beta III Tubulin knockout HAP1 whole cell lysate (20 µg)

Lanes 1 - 2: Merged signal (red and green). Green - ab18207 observed at 55 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab18207 was shown to recognize beta III Tubulin in wild-type HAP1 cells as signal was lost in beta III Tubulin knockout cells. An additional cross-reactive band at 50 kDa was observed in wild-type and knockout cells. Due to the immunogen’s homology with TUB (Tubby protein homolog, Uniprot: P50607), this lower band could correspond to the TUB protein. Please note that cross-reactivity with this protein has not been confirmed experimentally.

Wild-type and beta III Tubulin knockout samples were subjected to SDS-PAGE. Ab18207 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1 μg/ml and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature

ab18207 staining beta III Tubulin in SK-N-SH (Human 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 ab18207 at 1μg/ml and ab7291 at 1µg/ml overnight at +4°C, followed by a further incubation at room temperature for 1h with a Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) secondary antibody at 2 μg/ml (shown in green) and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) (ab150120) secondary antibody at 2 μg/ml (shown in pseudo color red). Nuclear DNA was labelled 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.

IHC image of ab18207 staining beta III Tubulin in rat 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 (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab18207, 1:2000 dilution, 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 haematoxylin 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.

Overlay histogram showing U-87MG (Human glioblastoma-astrocytoma epithelial cell line) cells stained with ab18207 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Triton X-100 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 (ab18207, 0.01μg/1x10⁶) for 30 min at 22ºC. The secondary antibody used was Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) at 1/4000 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (polyclonal) (ab171870, 0.01μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

This antibody gave a positive signal in U-87MG cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Triton X-100 for 20 min used under the same conditions.

ab18207 staining beta III Tubulin in Neuro-2a 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 ab18207 at 1μg/ml and ab7291 at 1µg/ml overnight at +4°C, followed by a further incubation at room temperature for 1h with a Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) secondary antibody at 2 μg/ml (shown in green) and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) (ab150120) secondary antibody at 2 μg/ml (shown in pseudo color red). Nuclear DNA was labelled 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.

ab18207 at 1/2000 staining mouse brain tissue sections by IHC-P. The tissue was formaldehyde fixed and an enzymatic antigen retrieval step was performed prior to incubation with the antibody for 16 hours. A biotinylated goat anti-rabbit IgG was used as the secondary.

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Overlay histogram showing Neuro 2A cells stained with ab18207 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Triton X-100 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 (ab18207, 0.01μg/1x10⁶) for 30 min at 22ºC. The secondary antibody used was Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) at 1/4000 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (polyclonal) (ab171870, 0.01μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

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 2% Bovine Serum Albumin before being incubated with ab18207 overnight at 4°C. Antibody binding was detected using Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody , and visualised using ECL development solution ab133406

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Immunohistochemistical staining (Formaldehyde/PFA-fixed paraffin-embedded sections) for Neuron specific beta III Tubulin antibody - Neuronal Marker (ab18207) on Dogfish/Catshark Tissue sections (head: snout region). Antigen retrieval step: Heat mediated. Blocking step: 1% BSA for 10 mins at RT. Primary Antibody used at 1/2000 incubated for 2 hours at RT. Secondary Antibody: Biotin labelled goat anti rabbit IgG (1/300).

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Differential expression of Dnmt1, Dnmt3a, and Dnmt3b during RA induced neuronal differentiation of P19 cells

Mouse P19 cells either left untreated (top panel) or RA treated for initial 2 days and further cultured for 4 days without RA (6 days, bottom panel) were immunostained with neuron specific β-III tubulin antibody and nuclei were stained using DAPI.

In order to confirm the neuronal morphology, the cells were stained for neuron specific beta III-tubulin (ab18207). RA induced P19 cells showed immunoreactivity against βIII-tubulin, indicating a neuronal phenotype. In contrast, undifferentiated P19 cells were βIII-tubulin negative.

(After Figure 1A of Sheikh et al)

Immunohistochemical analysis of adult mice ovaries undergone Clarity processing staining tyrosine hydroxlase (TH), Beta III Tubulin (Tuj1) with ab18207, and brain derived neurotrpic factor (BDNF) with ab72439. Positive staing of Tuj1 and BDNF is evident in the theca cells and corpus luteum.