BrdU protocol

Bromodeoxyuridine (BrdU) uses nucleotide substitution to replace thymidine with uridine in the DNA structure of dividing cells both in vitro and in vivo (Gage, 2000). BrdU has been utilised in a number of in vitro and in vivo studies to label a variety of cellular sources from human olfactory epithelium (Hahn et al., 2005) and neural progenitors (Nieoullon et al., 2005) to neural stem cells (Chu et al., 2004). BrdU antibody from Abcam Identification of the labelled cells can be applied both in vitro and in vivo to produce a nuclear staining pattern.

Protocol

• Tissue and cells are fixed with 4% paraformaldehyde in vitro for 30 mins at 4ºC, in vivo post fix for two hours at room temperature (r.t.)
• Following fixation, wash in 0.1M Phosphate Buffered Saline (PBS) (pH 7.4) with 1% TritonX100 (3x 5 mins)
• Incubate in HCl (1N) for 10 mins on ice to break open the DNA structure of the labelled cells
• This is followed by HCl (2N) for 10 mins at r.t before moving them to an incubator for 20 mins at 37°C
• Immediately after the acid washes, Borate buffer (0.1M) is added to buffer the cells for 12 mins at r.t.
• Samples are then washed in wash in 0.1M PBS (pH 7.4) with 1% TritonX100 (3x 5 mins) at r.t.
• Incubate in 0.1M PBS (pH 7.4) + 1% TritonX100 + Glycine (1M) + 5% normal goat serum (1hr) prior to incubating overnight (r.t) with anti-BrdU or a combination of anti-BrdU and other antibodies.
• Following the incubation overnight wash in 0.1M PBS (pH 7.4) with 1% TritonX100 (3x 5min)
• Samples can then be treated with a variety of secondary antibodies to visualise the anti-BrdU labelled cells (Fig 1 and Fig 2) including HRP conjugated secondaries with diaminobenzidine(DAB) or fluorescent conjugated antibodies such as AlexaFlour 488 or 533.

Tissue processing tips

In vivo and in vitro samples can be processed in this manner:

• Gelatin embedded tissue (cut between 30-50µm)
• OCT embedded tissue (cut between 10-30µm)
• Tissue culture cells

NB: The acid steps haven’t shown any adverse affect on the labelling of any other antigens tested so far in double labelling. Incubation times haven’t needed to be altered with thickness of section and everything is treated in the same manner

Examples

Fig. 1 BrdU protocol in vitro

In vitro dividing cells can be labelled with BrdU and following the protocol were visualised with HRP conjugated secondary antibodies and DAB to produce a nuclear staining pattern. Scale = 100µm

Fig. 2 BrdU protocol in vivo

In vivo cells can be labelled either by injecting BrdU or alternatively label the cells prior to transplantation and then identify them using the protocol. Neural stem cells were labelled 48hrs prior to transplantation into a spinal cord injury at cervical level 4 the cells were identified throughout the dorsal ventral axis of the cord and had also begun to migrate rostro caudally from injection site. They were visualised here using the protocol followed by a TRITC conjugated secondary antibody Scale = 500µm

Fig. 3 BrdU

In vivo cells were labelled prior to transplantation and then identified using Abcam Polyclonal anti-BrdU with a TRITC conjugated secondary antibody. Scale = 100µm

Reference List

Chu K, Kim M, Chae SH, Jeong SW, Kang KS, Jung KH, Kim J, Kim YJ, Kang L, Kim SU, Yoon BW (2004) Distribution and in situ proliferation patterns of intravenously injected immortalized human neural stem-like cells in rats with focal cerebral ischemia. Neurosci Res 50:459-465. Gage FH (2000) Mammalian neural stem cells. Science 287:1433-1438. Hahn CG, Han LY, Rawson NE, Mirza N, Borgmann-Winter K, Lenox RH, Arnold SE (2005) In vivo and in vitro neurogenesis in human olfactory epithelium. J Comp Neurol 483:154-163. Nieoullon V, Belvindrah R, Rougon G, Chazal G (2005) mCD24 regulates proliferation of neuronal committed precursors in the subventricular zone. Mol Cell Neurosci 28:462-474.

kindly provided by Daniel Webber, Neurorestoration Group, King's College London