ab6326 stained in Hela cells. Untreated and BrdU treated (10uM for 24 hours) cells were fixed with 100% methanol (5 min) and then subjected to acid hydrolysis using 2M HCL in 0.1% PBS-Tween for 30 minutes at room temperature to denature the DNA. They were then incubated with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% triton for 1h at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab6326 at 5µg/ml and ab7291 (Mouse monoclonal to alpha tubulin) at 1ug/ml overnight at +4°C. The secondary antibodies were Goat Anti-Rat IgG H&L (Alexa Fluor^® 488) preadsorbed (ab150165) (colored green) used at 2 ug/ml and Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) preadsorbed (ab150120) (pseudo-colored red) used at 1/1000 dilution for 1hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43µM for 1hour at room temperature.

ICC/IF image of ab6160 stained HeLa cells. The cells were 100% methanol fixed (5 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 (ab6160, 2µg/ml) overnight at +4°C. The secondary antibody (green) was ab150165 Alexa Fluor® 488 goat anti-rat IgG (H&L) used at 2µg/ml for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

Overlay histogram showing HeLa cells stained with ab19136 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween 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 (ab19136, 1μg/1x10^6 cells) for 30 min at 22ºC. The secondary antibody used was Alexa Fluor® 488 goat anti-rat IgG (H&L) at 1/2000 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rat IgG2a [aRTK2758] (ab18450, 2µg/1x10^6 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

Dot plot showing BrdU-treated HeLa cells stained with ab6326. Cells were incubated with 10 µM BrdU for 30 minutes prior to being harvested, washed twice in 1x PBS and fixed in 70% ethanol (4°C, added drop-wise) for at least 30 minutes on ice. Once fixed, pellets were acid denatured with 2M HCl for 30 minutes at 22°C and then neutralised with borate buffer (0.1M, pH8.5).
Samples were washed and incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab6326, 1µg/1x106 cells) for 30 min at 22°C. The secondary antibody used was Goat Anti-Rat IgG H&L (Alexa Fluor^® 488) preadsorbed (ab150165) at 1/2000 dilution for 30 min at 22°C.
7-AAD was added to cells 20 min prior to data acquisition.
Acquisition of >5,000 events were collected using a 50 mW Blue laser (488nm) with 530/30 and 685/35 bandpass filters.

Postnatal day 6 mouse testes were fixed with 4% paraformaldehyde. Tissue was embedded in O.C.T. and frozen. 5 micron sections were cut and transferred to slides. Sections were permeabilized with 0.1% Triton X-100 in PBS, and blocked with 3% BSA in 0.1% Triton X-100 + PBS. Sections were incubated with either (A) no primary antibody or (B ) anti-KIT (ab65525) for 1 h at RT. Sections were then washed 3X with 0.1% Triton X-100 in PBS and Goat-Anti Rat 488 (ab150165) applied at a 1/500 dilution. Sections were then mounted after washing 3X with 0.1% Triton X-100 in PBS.

HeLa cells showing negative staining by ICC/IF using only secondary antibody. The cells were 100% methanol fixed (5 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 secondary antibody (green) was ab150165 Alexa Fluor® 488 goat anti-rat IgG (H&L) used at 2µg/ml for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.