ICC/IF image of ab7750 stained HeLa cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab7750, 5µg/ml) overnight at +4°C. The secondary antibody (green) was anti-mouse DyLight® 488 (ab96879) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

Ab7750 staining human normal skin. Staining is localised to the cytoplasm.
Left panel: with primary antibody at 2 ug/ml. Right panel: isotype control.
Sections were stained using an automated system at room temperature. Sections were rehydrated and antigen retrieved with a retrieval buffer EDTA pH 9.0 . Slides were peroxidase blocked in 3% H₂O₂ in methanol for 10 minutes. They were then blocked for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 minutes and detected with an amplification kit for 30 minutes. Colorimetric detection was completed with diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin. Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.

Western blotting analysis labeling alpha-tubulin using ab7750 on lysates of various cell lines under reducing and non-reducing conditions. Nitrocellulose membrane was probed with 2 µg/ml of ab74696 followed by IRDye800-conjugated streptavidin. A specific band was detected for alpha-tubulin at approximately 54 kDa.

Overlay histogram showing HEK293 cells stained with ab7750 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Triton 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 (ab7750, 1µg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was anti-mouse DyLight® 488 (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HEK293 cells fixed with methanol (5 min)/permeabilized in 0.1% PBS-Triton used under the same conditions. 

Immunocytochemistry analysis of HeLa (human cervix carcinoma) cells labelling alpha tubulin (red) with ab7750. DAPI was used to stain the nuclei blue. 

Immunocytochemistry analysis of NIH 3T3 (mouse embryonal fibroblast) cells labelling alpha tubulin (green) with ab7750. DAPI was used to stain the nuclei blue.