Anti-BrdU antibody [BU1/75 (ICR1)] - BSA and Azide free

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-BrdU antibody [BU1/75 (ICR1)] - BSA and Azide free (AB264079)

ab6326 staining BrdU in Hela cells. Untreated and BrdU treated (10uM for 24 hours) cells. The 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 permeabilized with 0.1% PBS-Tween for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1hr. The cells were then incubated overnight at 4°C with ab6326 at 1μg/ml and ab7291 Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150165 Goat polyclonal Secondary Antibody to Rat IgG - H&L (Alexa Fluor^® 488) pre-adsorbed at 1/1000 dilution (shown in green) and ab150120 Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 594) pre-adsorbed 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS and Sodium Azide (ab6326).

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-BrdU antibody [BU1/75 (ICR1)] - BSA and Azide free (AB264079)

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 wasGoat 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 >5000 events were collected using a 50 mW Blue laser (488nm) with 530/30 and 685/35 bandpass filters.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-BrdU antibody [BU1/75 (ICR1)] - BSA and Azide free (AB264079)

IHC image of ab6326 staining in a formalin-fixed paraffin-embedded rat small intestine BrdU tissue section. The section was pre-treated using pressure cooker heat mediated antigen retrieval with sodium citrate buffer (pH6) for 30mins and incubated overnight at +4°C with ab6326 at 3 ug/ml. A goat anti-rat biotinylated secondary antibody was used to detect the primary and visualized using an HRP conjugated ABC system. The section was counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) users should optimize variable parameters such as antigen retrieval conditions primary antibody concentration and antibody incubation times.

This data was developed using the same antibody clone in a different buffer formulation containing PBS and Sodium Azide (ab6326).

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-BrdU antibody [BU1/75 (ICR1)] - BSA and Azide free (AB264079)

ICC/IF image of ab6326 stained HeLa cells both BrdU treated (left image) and normal cells (right image). The cells were 100% methanol fixed (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 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 (ab6326 10μg/ml) overnight at +4°C. The secondary antibody (green) was Goat Anti-Rat IgG H&L (DyLight^® 488) preadsorbed (ab98420) 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. Positive staining can be seen in the BrdU treated cells but not in the normal cells demonstrating specificity for BrdU.

This data was developed using the same antibody clone in a different buffer formulation containing PBS and Sodium Azide (ab6326).