[Histonet] brdU incorporation in keratinocytes

[Coolen, Neeltje]

Dear Histonetters,
 
I am trying to detect brdU incorporation in keratinocytes, but the
keratinocytes which are not pulsed with brdU also stained positive for
brdU. This was the protocol I used:
The cells were pulsed with medium containing 100uM brdU (Sigma, cat. no.
B5002) or with medium alone for 3h, fixed in 4% formaldehyde at 4oC for
30 min, incubated in HCl (1N) for 10 min on ice followed by incubation
in HCl (2N) for 10 min at r.t before moving them to an incubator for 20
min at 37oC. Immediately after the acid, cells were washed in 0.1 M
sodium borate (pH 8.5) for 10 min, incubated with 5% normal rabbit serum
for 30 min and incubated with anti-brdU antibody (1:50, MP Biomedicals,
clone IIB5) for 1h at r.t. As a secondary antibody I used biotinylated
rabbit anti-mouse Ig (DAKO) and  I used streptavidine alexa fluor 488
(Molecular Probes).
 
Does anyone have a solution for this problem?
 
Thank you for your help.
 
Neeltje Coolen
 
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N.A. Coolen, PhD student
VSBN
Zeestraat 26
1941 AJ Beverwijk
The Netherlands
 
tel: +31 251 275 564