Double staining NeuN and beta-amyloid

[amelie]

Hi. I am trying to detect simultaneously NeuN and beta-amyloid in mice cerebral cortex. Both anti-NeuN and anti-beta-amyloid are mouse IgG (I could not find them monoclonal from other species, only polyclonal). I want to use AlexaaFlour-conjugated secondary antibodies, however, I am not sure if I can actually distinguish them because they are both mouse IgG. Is this right? Any suggestions on how I could get around this would be very very welcome. Thanks in advance!

Amelie

[CanuckPhD]

Are you using frozen or paraffin sections? Do you want to look at plaques or amyloid itself? What is your over all goal (example look for neuronal numbers near plaques or neurons containing amyloid)?Give us a few more details and we should be able to help you out.

[amelie]

Thanks for your reply. I am using paraffin embedded samples. My main goal is to see whether there are neurons containing amyloid after exposing the animals to metal particles. The protocol I was given just said use NeuN as a neuronal marker. The problem is that I dont think I can detect both with the secundary antibody since they ar both mouse IgG. Do you think I can determine amyloid with the need of the neuronal marker? Thanks again,

Amelie

[amelie]

Could I use a rabbit polyclonal for NeuN?

[CanuckPhD]

We have had success microwaving between the two antibodies. We stain with the first, in this case likely NeuN then fluorescent secondary, microwave (citrate buffer pH6.0 15 minutes), then do second primary (amyloid). There is little binding of the second secondary to the first primary due to microwave induced changes to the first antibody.

Reference: Toth and Mezey Journal of Histochemistry and Cytochemistry 55:545-554 2007

[Thitus]

Hi Amelie,

I had a similar problem. The issue is that there are no polyclonal rabbit NeuN antibodies available (at least to my knowledge). For double-staining using Abs from the same host species you could use the Invitrogen Zenon staining kit. It provides you with different fluorescent Fab fragments that bind to your primary antibodies. The section thickness is limited to 12-14um.

http://www.invitrogen.com/site/us/en/home/References/Molecular-Probes-The-Handbook/Antibodies-Avidins-Lectins-and-Related-Products/Zenon-Technology-Versatile-Reagents-for-Immunolabeling.html

Hope this helps.


Should you come across a non-mouse polyclonal NeuN-AB let me know.

Thitus

[szatmar]

Aside from your technical problem, do you use transgenic animals carrying the human APP mutant protein or you just want to see the effect of metal toxicity on amyloid processing in wild-type? In a later case, you would never get Abeta staining since mice do not process APP in the way human do. Just in case...