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).

 

Lanes 1 & 3 : Anti-Tubulin antibody [YL1/2] - Loading Control (ab6160) at 1/5000 dilution
Lanes 2 & 4 : Anti-Tubulin antibody [YL1/2] - Loading Control (ab6160) at 1/10000 dilution

Lanes 1-2 : HeLa (Human epithelial carcinoma cell line) whole cell lysate
Lanes 3-4 : BALB/3T3 whole cell lysate (ab7901)

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Rabbit Anti-Rat IgG H&L (HRP) (ab6734) at 1/2000 dilution

Predicted band size: 50 kDa
Observed band size: 52 kDa

why is the actual band size different from the predicted?
Additional bands at: 17 kDa, 34 kDa, 80 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 10 seconds

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.

All lanes : Anti-Tubulin antibody [YL1/2] - Loading Control (ab6160) at 1 µg/ml

Lane 1 : HeLa (Human epithelial carcinoma cell line) whole cell lysate
Lane 2 : NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate
Lane 3 : PC-12 (Rat adrenal pheochromocytoma cell line) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Peroxidase Conjugated Rabbit Anti-Rat IgG (H+L) at 1/10000 dilution

Performed under reducing conditions.

Predicted band size: 50 kDa
Observed band size: 52 kDa why is the actual band size different from the predicted?
Additional bands at: 85 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 8 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.