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BrdU IHC-Bizbenzimide problem


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#1 smr86

smr86

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Posted 08 June 2012 - 05:47 AM

Hi there,

I've recently begun staining GFP mouse brain sections (PFA-perfused, followed by 24h post-fixation, and cryoprotection in 30% sucrose, snap frozen in isopentane and sectioned onto gelatin-coated slides using the cryostat set to 40um) for BrdU as well as neuronal and NPC markers. Initially, I did a 20min antigen retrieval step (0.01M sodium citrate pH6 at 85C) followed by (in the case of sections for BrdU staining only) a 30min 2N HCl incubation. This gave me really nice specific staining for BrdU and all my other neural markers, but the GFP fluorescence was non-existant.

Having read that antigen retrieval can cause problems with GFP fluorescence, I repeated the test this time omitting the sodium citrate test, and still got really nice images and a little bit of an improvement on the GFP fluorescence so I will now use a GFP antibody to hopefully increase my signal.

What I have noticed however is a problem with the bizbenzimide staining. Sections that have not been treated with 2N HCl have perfect nuclear staining; sections that have been treated with 2N HCl have very poor nuclear fluorescence, almost background staining like in appearance. This is creating a bit of a problem for me as the sections have to be treated with the HCl for BrdU staining, I did try staining sections with no HCl treatment and got terrible BrdU results (as expected).

Does anyone have any experience with this or have any suggestions? The nuclei do appear normal in the section, but they are just not really of any use for my analysis in their current dull state.

Thanks in advance Posted Image




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