Anti-BrdU antibody [BU1/75 (ICR1)] - Proliferation Marker (ab6326)

(A) Schematic of BrdU pulse labeling. (B) E15.5 whole brain section incorporated with BrdU after IUE at E13.5. Dashed rectangle indicates cortical area for quantification. Scale bar, 200 μm. (C) BrdU incorporation in mice subjected to IUE with indicated plasmids. Scale bar, 50 μm.

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.

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 22ºC 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 22ºC.

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.

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Left panel: Visualization of cell division using bromodeoxyuridine (BrdU). Antibodies recognizing NeuN in blue mark nuclei of mature neurons, antibodies recognizing Dcx are in red and antibodies against BrdU are in green. Scale bar: 50 µm. Right panel: Quantification of the BrdU⁺ cells after 4 days. Rev-erbα^−/− mice display more BrdU positive cells (mean ± SEM, n = 3, **p<0.005, t-test).

Schnell A et al., PLoS One 9(6): e99883, fig 5b. Doi: https://doi.org/10.1371/journal.pone.0099883.

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.

SPF mice (4-week-old) were provided with drinking water supplemented (or not) with an antibiotic cocktail (Abx: ampicillin, vancomycin, metronidazole, neomycin) for 4 weeks, after which frozen sections of the ileum from mice (8-week-old) were prepared at 2 h or 2, 3, or 5 days after BrdU injection and were immunostained with mAbs to BrdU (red) and to β-catenin (green). The boxed areas in the left panels for the sections prepared at 2 h after BrdU injection are shown at higher magnification in the right panels. Scale bars, 20 μm (higher magnification) or 100 μm (lower magnification).

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.

ab6326 staining BrdU in mouse brain (dentate gyrus) tissue sections by Immunohistochemistry (PFA perfusion fixed frozen sections). Tissue samples were perfused with 1X PBS followed by 4% paraformaldehyde and then cryopreserved in 20-30% sucrose. 20-25 µm sections were permeablized with 1% Triton X-100 + 0.5% Tween 20 in 1X PBS. Sections were treated with 1 N HCL for 10 min followed by 2 N HCL for 10 min at RT and then 20 min at 37°C. Then sections were incubated with borate for pH correction and permeablized with 1 X TBS and blocked with 3-5% donkey serum.  Samples were incubated with the primary antibody (1/1000 in 1X PBS + 0.1% Tween 20) at 4°C overnight. Donkey Anti-Rat IgG H&L (Alexa Fluor® 568) preadsorbed (ab175475) (1/250) was used as the secondary antibody.

Green - DCX.
Red - BrdU.
Blue - NeuN.

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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.

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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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