Anti-Tubulin antibody [YL1/2] - Loading Control (ab6160)

Cytoskeleton and major extracellular matrix proteins in human DPSC (Dental pulp stem cell) were analyzed by immunofluorescence.

Vimentin and tubulin (Panel D, control, E, (BD) and F, (BR)) are shown.

After 7 days in contact with/without the materials (Biodentine (BD) and Bioroot (BR)), coated coverslip cultures were fixed in PBS (pH 7.4) containing 4% paraformaldehyde/5% sucrose for 10 minutes. For detection of intracellular molecules, the cells on the coverslips were permeabilized using 0.5% Triton X-100. To block background staining, cells were treated with PBS containing 1% BSA/1% glycine at 37°C for 20 minutes. Samples were incubated with the primary antibody at 4°C overnight or at 37°C for 2 hours. For double immunostaining, primary antibodies were incubated as above. Samples were then incubated with the appropriate secondary antibodies at 37°C for 1 hour. Cell nuclei were stained using DAPI.

Scale Bar: 100 μm.

Egr3 localization is associated with microtubule organization in mouse oocytes.

Mouse oocytes stained for Tubulin using ab6160 (Right panels, red) in ICC/IF.

The localization of Egr3 and microtubule at thawing of vitrified oocytes. Vitrified MII oocytes were stored in LN₂ for 2 weeks. Oocytes were taken out from LN₂, incubated in decreasing concentrations of sucrose, and then fixed immediately. These oocytes were subjected to immunofluorescence staining with anti-Egr3 and ab6160 antibodies. Arrows indicate the growing arrays of microtubules at the site of Egr3 accumulation.

Green: Egr3.

Red: Microtubule (MT).

ICC/IF image of ab6160 stained HeLa (Human epithelial cell line from cervix adenocarcinoma) cells.

The cells were fixed in 100% methanol 5 minutes, permeabilized with 0.1% Triton X-100 for 5 minutes and then incubated in 1% BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1 hour to block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab6160, 1/1000 dilution) overnight at +4°C. The secondary antibody (green) was ab150165 Alexa Fluor^® 488 goat anti-rat IgG (H+L) pre-adsorbed, used at a 1/1000 dilution for 1 hour. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

The negative control (inset) is a secondary-only assay to demonstrate low non-specific binding of the secondary antibody.

This product also gave a positive signal under the same testing conditions in HeLa cells fixed with 4% formaldehyde (10 minutes).

IHC image of Tubulin staining in human colon formalin fixed paraffin embedded tissue section*, performed on a Leica Bond™ system using the standard protocol F.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer pH 6 for 20 minutes. The section was then incubated with ab6160, 5 µg/ml, for 15 minutes 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.

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.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

Overlay histogram showing HeLa (Human epithelial cell line from cervix adenocarcinoma) cells stained with ab6160 (red line).

The cells were fixed with 80% methanol (5 minutes) and then permeabilized with 0.1% PBS-Tween for 20 minutes. 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 (ab6160, 1 µg/1x10⁶ cells) for 30 minutes at 22ºC. The secondary antibody used was DyLight^® 488 goat anti-rat IgG (H+L) (ab98386) at 1/500 dilution for 30 minutes at 22ºC. Isotype control antibody (black line) was rat IgG2a [aRTK2758] (ab18450, 1 µg/1x10⁶ cells) used under the same conditions.

Acquisition of >5,000 events was performed.

ab6160 staining Tubulin in mouse trophoblast giant cells by ICC/IF (Immunocytochemistry/Immunofluorescence).

Cells were fixed with methanol and blocked with 0.5% BSA for 30 minutes at 20°C. Samples were incubated with primary antibody (0.5% BSA, 0.1% Tween-20 PBS) for 1 hour at 20°C. An Alexa Fluor^® 568 polyclonal Goat anti-Rat IgG (H+L) Cross-Adsorbed (1/750 dilution) was used as the secondary antibody.

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ab6160 staining Tubulin in mouse MEF cells by Immunocytochemistry/ Immunofluorescence.

Cells were fixed with 2% PFA  and 96% Ethanol. Samples were incubated with primary antibody (1/2000 in 0.1% Saponin/1% BSA/PBS) for 1 hour. A Cyt3^®-conjugated goat polyclonal to rat IgG (H&L) was used at dilution at 1/500 as secondary antibody.

Red staining in the image represents Tubulin, whereas the green one resembles gamma-tubulin.

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ab6160 staining mouse prostate tissue sections by IHC-P.

The tissue was serial sectioned at 6 microns, formaldehyde fixed and subjected to heat mediated antigen retrieval prior to blocking in 3% peroxidase for 5 minutes at 27°C. The primary antibody was diluted 1/500 and incubated with the sample for 16 hours at 4°C. A Cy5^® conjugated goat anti-rat antibody was used as the secondary.

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ab6160 at 1/1000 dilution staining Tubulin in human WBC cells by Immunocytochemistry/ Immunofluorescence.

Cells were fixed in acetone and then blocked in 5% serum for 1 hour at 25°C. No permeabilization was done. The primary antibody was used at 1/1000 dilution in PBS-Tween and incubated with sample at 4°C for 16 hours. An Alexa Fluor^® 594 conjugated goat polyclonal to rat IgG was used as secondary at 1/500 dilution.

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This image was kindly supplied as part of the review submitted by Marko Kallio. ab6160 was used for immunofluorescence on male rat testis samples in order to visualize microtubules of meiotically deviding cells. The samples were fixed with 2% paraformaldehyde and 0.8% glutaraldehyde and the antibody was used at a dilution 1:2500 (red - tubulin, blue - DNA stained with DAPI).

ab6160 at a 1/200 staining Tubulin in mouse liver tissue sections by Immunohistochemistry (frozen sections) incubated for 9 hours at +4°C. Fixed in formaldehyde, permeabilized using 0.2% Triton X-100. Blocked using 2% BSA for 30 minutes at 20°C. Secondary used at a 1/200 dilution polyclonal Goat anti-rat IgG conjugated to Alexa Fluor^® 555.

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