ab6326 stained inHela cells. Untreated andBrdU treated (10uM for 24 hours)cells were fixed with 100% methanol (5 min) and then subjected to acid hydrolysis using 2M HCLin 0.1% PBS-Tween for 30 minutes at room temperature to denature the DNA. They were thenincubated 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 andab7291 (Mouse monoclonal toalpha tubulin) at 1ug/ml overnight at +4°C. The secondary antibodies wereGoat Anti-Rat IgG H&L (Alexa Fluor® 488) preadsorbed (ab150165)(colored green) used at2 ug/ml andGoat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (ab150120)(pseudo-colored red) used at1/1000 dilutionfor 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.

Dot plotshowing BrdU-treated HeLa cellsstained 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 at22ºC and then neutralised with borate buffer (0.1M, pH8.5).

Samples were washed andincubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab6326, 1µg/1x10⁶ cells) for30 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 22ºC.

7-AAD was added tocells 20 min prior to data acquisition.

Acquisition of >5,000 events were collected using a 50 mW Blue laser (488nm) with530/30 and 685/35bandpass filters.

Confocal immunofluorescence sections (~0.3 μm thick) showing the localization of the different factors in HEK 293 cells infected with Ad5 wt. Double labeling for BrdU (ab6326, magenta) and L1 52/55 kDa (green). Dotted rectangles indicate the areas shown at higher magnification in the bottom row. Bars: 3 μm.

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) wasGoat 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.

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 at3 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.

ab6326 staining cultured cells of rat brain tissue by ICC.  The sample was PFA fixed and permeabilized in 1M HCl prior to blocking with 5% serum for 1 hour at 25°C.  The primary antibody was diluted 1/500 and incubated with the sample for 16 hours at 25°C.  A biotinylated rabbit anti-rat IgG antibody, diluted 1/200, was used as the secondary.

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ab6326 staining BrdU in mouse lung tissue sections by Immunohistochemistry (Formalin/PFA-fixed parafin-embedded sections). Tissue samples were heat mediated withCitrate buffer for 40min.The sections were blocked with 10% donkey serum for 1 hour at 22°C.Samples were incubated with the primary antibody (1/200 in 10% Donkey serum-PBS 0.2% Triton) at 4°C for 16 hours. Cy™3 AffiniPure Donkey Anti-Rat IgG (H+L)(1/400) was used as the secondary antibody.

Representative confocal images of terminal bronchioles in mouse lungs taken at day 5 following naphthalene injection.

Colors label CC10 (red), FoxJ1 (green), BrdU (yellow), and nuclear DNA (blue).

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