Anti-beta III Tubulin antibody [2G10] - Neuronal Marker (ab78078)

Soft palate innervation is primarily oro-medial and complements the differentiated muscle pattern

(A-L) MHC (green) and β3-tubulin (red) co-immunostaining along the antero-posterior axis of the mouse soft palate at E13.5 (A-C), E14.5 (D-F), E16.5 (G-I), and P0 (J-L) at the level of the TVP and PLG (A, D, G, J), LVP (B, E, H, K), and PLP and SPC (C, F, I, L). Dashed lines indicate the outline of the palatal shelf and tongue epithelium. Arrows indicate the nerve fibers innervating the palatal shelves in a pattern complementary to the differentiated muscle, mainly located orally in the medial region. The schematic drawings indicate the orientation and the position of each section. Scale bars: 200μm.

beta III Tubulin is detected with ab78078.

(From Figure 4 of Grimaldi et al)

ab78078 staining beta III Tubulin (red) in mouse differentiated neural stem cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with 0.2% Triton X-100. Samples were incubated with primary antibody (5µg/ml in PBS + 3% BSA) for 16 hours at 4°C. An Alexa Fluor^® 568-conjugated donkey anti-mouse IgG polyclonal (1/1000) was used as the secondary antibody. Blue - DAPI nuclear counterstain.

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Lane 1: Wild-type HAP1 whole cell lysate (20 µg)
Lane 2: Beta III Tubulin knockout HAP1 whole cell lysate (20 µg)
Lane 3: HeLa whole cell lysate (20 µg)
Lane 4: Hek293 whole cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab78078 observed at 50 kDa. Red - loading control, ab181602, observed at 37 kDa.

ab78078 was shown to specifically react with beta III Tubulin in wild-type HAP1 cells as signal was lost in beta III Tubulin knockout cells. Wild-type and beta III Tubulin knockout samples were subjected to SDS-PAGE. ab78078 and ab181602 (Rabbit anti-GAPDH loading control) were incubated overnight at 4°C at 10 μg/ml and 1/20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed ab216772 and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed ab216777 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

IHC image of ab78078 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 6.0 epitope retrieval solution 1) for 20 mins. The section was then incubated with ab78078, 0.5μg/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 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.

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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 ab78078 overnight at 4°C. Antibody binding was detected using an anti-mouse antibody conjugated to HRP, and visualised using ECL development solution ab133406

ab78078 staining beta III Tubulin in NGF-differentiated PC12 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 ab78078 at 1μg/ml 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 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 ab78078 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 EDTA/Tris (epitope retrieval solution 2) for 20 mins. The section was then incubated with ab78078, 0.5μg/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 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.

Immunohistochemistical detection of Neuron specific beta III Tubulin antibody using ab78078 on formaldehyde-fixed paraffin-embedded marmoset cerebellum sections. Antigen retrieval step: Heat mediated in Citric acid pH6 buffer. Permeabilization: No. Blocking step: 1% BSA for 10 mins @ rt°C. Primary Antibody used at 1/1000 incubated for 2 hours in TBS/BSA/azide. Secondary Antibody: anti mouse IgG conjugation to biotin (1/200).

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Immunohistochemistical detection of Neuron specific beta III Tubulin using antibody (ab78078) on formaldehyde-fixed paraffin-embedded human medulla oblongata sections. Antigen retrieval step: Heat mediated in citric acid HIER buffer. Permeabilization: No. Blocking step: 1% BSA for 10 mins @ rt°C. Primary antibody dilution 1/250 incubated for 2 hours in TBS/BSA/azide. Secondary antibody: anti Mouse IgGs conjugated to biotin (1/200). This section was cut from an anonymous autopsy P.wax block that is over 20 yrs old, so I would expect variable positivity when compared with more recently sampled tissues (giving higher dilution factors, such as I obtained with fresh mouse/rat CNS blocks.) Transverse-cut axons stain very intensely longitudinal nerve processes and cell bodies are not so heavily stained but are still easily seen.

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ab78078 staining beta III Tubulin in Chicken e6: developing eye tissue sections by Immunohistochemistry (IHC-P - formaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with 1% BSA for 10 minutes at room temperature; antigen retrieval was by heat mediation in a citrate buffer. Samples were incubated with primary antibody (1/1250) for 2 hours. A Biotin-conjugated Goat anti-mouse IgG polyclonal (1/200) was used as the secondary antibody.

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