Does anyone have experience with Mouse Anti BrdU Immuno protocol?
I am working on young rat's brains exposed to prenatal BrdU (i.p.).
Literature available are confusing.
Will I be required to carry out antigen retrieval considering that the brain samples could be said to be over-fixed, as they stayed in 4% PFA for 48 hours?
We have had great results with an adapted protocol of the BD BrdU In-situ kit, maybe you can take this manual as a guideline. We use DAKO AG retrieval solution (25`in a steamer), basically it is a citrate buffer.
http://www.bdbiosciences.com/us/applications/research/apoptosis/cell-cycle-and-proliferation-kits-sets/brdu-iin-situi-detection-kit/p/550803
I used BrdU antibody (clone G3G4) from DSHB (http://dshb.biology.uiowa.edu/G3G4AntiBrdUrd ) to get excellent results.
the cryosections, with 10~15micro on coated slides, pre-treated with bouin-4 for 15min, antigen retrieval by putting slides in citrate buffer pH6.0 in a boiling water bath for 10min.
no HCl treatment needed. you can ref the following protocol sggested by Tang et al (http://www.jneurosci.org/content/27/22/5837.full) .
Thanks Victoria and Hongyun for the provided information. From the literature links I can easily work something out.
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