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

• Flow Cyt (Intra)

AbReview29284****

Flow Cytometry (Intracellular) - Anti-BrdU antibody [BU1/75 (ICR1)] - Proliferation Marker (AB6326)

ab6326 staining BrdU in HeLa cells by Flow Cytometry. Cells were incubated with 10 µM BrdU for 30 minutes prior to being harvested with 1X trypsin-EDTA washed twice in PBS containing 1% BSA and fixed in 70% ethanol (added drop-wise) for at least 30 minutes on ice. Once fixed pellets were acid denatured with HCl/Triton X-100 for 30 minutes at room temperature and then neutralised with sodium tetraborate.
Pelleted cells were re-suspended in Tween/BSA/PBS to which primary antibody was then added (0.1 µg in 0.5% Tween 20 (v/v) plus 1% BSA in PBSA) and incubated for 30 minutes at room temperature. Secondary Alexa Fluor^®488-conjugated Goat anti-Rat IgG (H+L) was used at 1/500 and incubated for 30 minutes at room temperature in the dark. Cells were pelleted once more and resuspended in PBS containing 5 µg/mL propidium iodide.
Gating Strategy : Based on forward and side scatter cells were gated into the region used for analysis.

This image is courtesy of an anonymous Abreview

• IHC-P

AbReview70639****

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-BrdU antibody [BU1/75 (ICR1)] - Proliferation Marker (AB6326)

ab6326 staining BrdU in mouse lung tissue sections by Immunohistochemistry (Formalin/PFA-fixed parafin-embedded sections). Tissue samples were heat mediated with Citrate 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 dilution) 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).

This image is courtesy of an anonymous Abreview

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-BrdU antibody [BU1/75 (ICR1)] - Proliferation Marker (AB6326)

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.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-BrdU antibody [BU1/75 (ICR1)] - Proliferation Marker (AB6326)

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.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-BrdU antibody [BU1/75 (ICR1)] - Proliferation Marker (AB6326)

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)] - Proliferation Marker (AB6326)

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.

• IHC-P

AbReview10196****

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-BrdU antibody [BU1/75 (ICR1)] - Proliferation Marker (AB6326)

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.

This image is courtesy of an anonymous Abreview

• IHC-P

PubMed

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-BrdU antibody [BU1/75 (ICR1)] - Proliferation Marker (AB6326)

Immunohistological staining of frontal (A-C) and sagittal (D) sections through a tooth family on the lower jaw of S. salar with BrdU labelled cells in green and DAPI counterstain in blue. The tooth families presented are in a similar state of development : a young functional tooth and its successor in morphogenesis stage. (A) Pulsed specimen, T0; (B) Chase time T1, one week after BrdU administration; (C) Chase time T2, two weeks after BrdU administration and (D) Chase time T3, four weeks after BrdU administration. The white arrow indicates single cells within the middle dental epithelium (MDE) enclosed by the IDE, cervical loop and ODE of the replacement tooth. Abbreviations : Ab : aboral; Ant : anterior; CV : cervical loop; DP : dental papilla; FT : functional tooth; IDE : inner dental epithelium; Lab : labial; Lin : lingual; MDE : middle dental epithelium; ME : mesenchyme; OE : oral epithelium; ODE : outer dental epithelium; Or : oral; PC : pulp cavity; Pos : posterior; yellow asterisk : replacement tooth; scale bars : 100 μm.

Immunohistological staining for BrdU on the paraffin sections : Rehydration through a decreasing ethanol series, chromatin precipitation in hydrochloric acid, block in 3% BSA/ 1% milk powder, exposure to primary antibody (ab6326) and secondary antibody (polyclonal anti-rat Alexa Fluor^® 488), DAPI counterstaining (1μl/ml). Immunofluorescence was visualized on a NIKON eclips TE2000-S confocal laser-scanning microscope. Adobe Illustrator CS5, Adobe Photoshop CS5 and Fiji were used to process bright field and fluorescent images of the immunostained sections.

Image from Vandenplas S et al., PLoS One. 2016;11(4):e0152870. Fig 2.; doi: 10.1371/journal.pone.0152870. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/