Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - 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)

Grimaldi, A. et al PLoS One. 2015 Dec 15;10(12):e0145018. doi: 10.1371/journal.pone.0145018. eCollection 2015. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/.

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

Neuron specific beta III Tubulin - Neuronal Marker was immunoprecipitated using 0.5mg Mouse Brain whole tissue lysate, 5µg of Mouse monoclonal [2G10] to Neuron specific beta III Tubulin - Neuronal Marker and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).

The antibody was incubated under agitation with Protein G beads for 10min, Mouse Brain whole tissue lysate lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.

Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab78078.

Secondary: Goat polyclonal to mouse IgG light chain specific (HRP) at 1/5000 dilution.

Band: 50kDa: Neuron specific beta III Tubulin - Neuronal Marker.

All lanes:

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

Predicted band size: 50 kDa

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

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.

All lanes:

Western blot - Anti-beta III Tubulin antibody [2G10] - Neuronal Marker (ab78078) at 1/20000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

Beta III Tubulin knockout HAP1 whole cell lysate at 20 µg

Lane 3:

HeLa whole cell lysate at 20 µg

Lane 4:

HEK-293 whole cell lysate at 20 µg

Predicted band size: 50 kDa

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

ab78078 staining beta III Tubulin in NGF-differentiated PC12 cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Triton X-100 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 ab78078 at 1µg/ml and ab6046, Rabbit polyclonal to beta Tubulin - Loading Control. Cells were then incubated with ab150117, Goat polyclonal Secondary Antibody to Mouse IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (shown in green) and ab150080, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 594) 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.