BrdU labeling in primary culture - (May/09/2007 )
Hi,
I am not sure how long to incubate my neurons with BrdU as methods seem to vary massively. I am also torn between using fluorescent labelling or DAB. Does anyone have a tried and tested protocol for this? I am struggling to find anything on the web specific to neurons.
Cheers!
-Hellie-
QUOTE (Hellie @ May 9 2007, 09:06 AM)
Hi,
I am not sure how long to incubate my neurons with BrdU as methods seem to vary massively. I am also torn between using fluorescent labelling or DAB. Does anyone have a tried and tested protocol for this? I am struggling to find anything on the web specific to neurons.
Cheers!
I am not sure how long to incubate my neurons with BrdU as methods seem to vary massively. I am also torn between using fluorescent labelling or DAB. Does anyone have a tried and tested protocol for this? I am struggling to find anything on the web specific to neurons.
Cheers!
The duration of the BrdU pulse does vary widely, from as little as a few hours to a few days! If you can't find anything in the literature (did you check PubMed?), the best thing to do is test a few different pulse lengths in your positive control samples and see what gives the level of signal you want. Molecular Probes (now part of Invitrogen) has a great anti-BrdU-Alexa647 antibody.
Other crucial things with this technique are your permeabilization technique (there are a couple) and the length of time you digest with DNAse.
Good luck!
-Cassio-