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Directly conjugated antibodies for immunofluorescence
All tags IHC protocols BrdU staining protocol
Bromodeoxyuridine (5-bromo-2-deoxyuridine, BrdU) is a synthetic analog of the nucleoside thymidine. It is incorporated into replicating DNA in dividing cells and can be subsequently detected by anti-BrdU antibodies.
Immunostaining of the labeled cells is a standard IHC/ICC procedure but requires a step to denature DNA, allowing the antibody access to the BrdU. The procedure can be applied to cell culture as well as tissue sections. Hydrogen chloride is a popular choice for the denaturing agent. The following procedure is an abbreviated version of the protocol linked on the datasheet for BrdU antibody Proliferation Marker (ab1893), provided courtesy of D. Webber.
Hydrogen chloride (HCl), 1 M and 2 M
Borate buffer 0.1 M
Borax (sodium tetraborate) 38.1 g/liter, pH to 9.0
Phosphate-buffered saline (PBS), pH 7.4, with 0.1% Triton X-100
The acid incubation may affect the labeling of other antigens in multiple labeling; this will depend on the antigen and the antibody used to label it. Campana et al. (1988), discusses an alternative to the above procedure for double and triple staining of BrdU, Ki67, and TdT using NaOH instead of HCl to denature DNA.
Another modification, used in Matsuura et al. (1997), includes digestion of nuclear protein in resin-embedded sections. Sections were treated with 2 M HCl at 60°C for 15 min and washed in water for 15 min. After dehydration in an ethanol series, the sections were etched with xylene (15 min, twice), digested with 0.025% protease in PBS at 37°C for 10 min, and washed with cold PBS 3 times for 5 min.
Fig 1: BrdU protocol in vitro
In vitro dividing cells can be labeled with BrdU and following the protocol were visualized with HRP conjugated secondary antibodies and DAB to produce a nuclear staining pattern. Scale bar = 100 µm.
Fig 2: BrdU protocol in vivo
In vivo cells can be labeled either by injecting BrdU or alternatively label the cells prior to transplantation and then identify them using the protocol. Neural stem cells were labeled 48 hr prior to transplantation into a spinal cord injury at cervical level 4. The cells were identified throughout the dorso-ventral axis of the cord and had also begun to migrate rostro-caudally from the injection site. They were visualized using the described protocol, followed by a TRITC conjugated secondary antibody. Scale bar = 500 µm.
Fig 3: BrdU protocol in vivo
In vivo cells were labeled prior to transplantation and then identified using Abcam Polyclonal anti-BrdU with a TRITC conjugated secondary antibody. Scale = 100 µm.
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