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Thread: BrdU Labeling

  1. #1 BrdU Labeling 
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    Dear all,I have a big problem with the "hard" staining with the Ab anti- BrdU (sigma) in peroxydase but nothing is going well...The samples are brains rats and they were sacrificed after 7 days from the 1st BrdU i.p. injection...the fixation is characterized by 10 in in PFA 4%, and then denaturation with Hcl 2M 37C 30min and neutralize it with Sodium borate 0.1M 10min RT. I tryed with different concentration but...nothing (1/10, 1/100, 1/200 in blocking solution 4C o/n).Thank you in advance for your suggestions!!!Mimi


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  3. #2  
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    Hi Mimikundera,

    Well, I have no personal experience with BrdU labelling, I was aware that it provides a readout of cellular proliferation but I did not know that it was possible to administer BrdU in vivo. Anyhow, I will try to help as best I can.

    Firstly, are you sure that the IP injection itself is working? I.e. the person performing it is experienced and is injecting into the right place? Alternatively, is it possible to try administering the BrdU by another method e.g. IV injection?

    Also, is an incubation time of 7 days normally used in the field? Invitrogen suggest an incubation time of just 2 hours:

    'Labeling with BrdU In Vivo

    1. Inject the animal with labeling reagent. As a general rule, 1 ml concentrated reagent per 100 g body weight is a suitable amount. Intraperitoneal injections are recommended for mice.
      Note: For larger animals use 10 ml per 1 kg of body weight.
    2. Wait 2 hours and sacrifice the animal. Remove the organs you wish to study.
    3. Process the tissue as necessary (frozen sections or paraffin embedding).
    4. Cut sections for immunohistochemistry.'


    From BrdU Labeling Protocol

    Also, how penetrable is the blood-brain barrier to BrdU? Could this be cauing an uptake issue?

    If you haven't already done so, it might also be worth taking a look at Nowakowski, Lewin and Miller's 1988 Brain Cell Biology paper in which they establish the method for BrdU labelling of brain tissue. Unfortunately, I can only get my hands on the abstract as opposed to the methods section, but hopefully you will have better luck: SpringerLink - Journal of Neurocytology, Volume 18, Number 3
    Are the fixation and denaturation conditions that you are using standard, tried-and-tested protocols? Maybe try a 1/1000 solution? (Just a stab in the dark - 1/1000 is generally a good benchmark concentration when experimenting with a new Ab!). Could you try incubating for longer than overnight? Presumably, you are using some commercial products... what are their provided guidelines?

    Finally - have you tried asking your supervisor and/or other Lab members who might already have experience with the method?

    BW,

    Tridimity.






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  4. #3  
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    Thank you very much!
    Your advises are very helpful!!!
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  5. #4  
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    Also, are you sure that your Ab hasn't gone off?

    BW,

    Tri~
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