Hello,

First, you should appreciate the fact that the central aim of this experiment, from the information you gave, is to investigate any DIFFERENTIAL hyperphosphorylation of Tau in the 3x-Tg mouse compared to the wild type control (you said "healthy"). If this is the case, then you cannot objectively obtain any differential outcome (Tau hyperphosphorylation) while using a different parameter (different anti-phosphoTau antibodies). Therefore, you should use the same anti-phosphoTau antibody for both control and 3x-Tg brain slices.

Having said this, you must also appreciate the fact that Tau phosphorylation is SITE-SPECIFIC and so is the anti-phosphoTau antibody. So, different anti-phosphoTau antibodies recognize different phosphosites, which anti-phosphoTau antibody to use depends on YOUR phosphosite of interest. Then, regarding inter-compartmental staining of Tau, please understand that Tau compartmentalization is quite different between wildtype control and Alzheimer's disease (AD) model, because there is Tau mislocalization from the axon into the dendrites in AD. The mechanism of Tau mislocalization is still being worked out, but suffice to say that in wild type control, anti-total Tau antibody (not anti-phosphoTau antibody please) will stain axonal Tau, but the same anti-total Tau antibody will stain dendritic Tau in AD model mice. So, for both control and AD mice brain slice, you should combine the axonal marker (anti-Tau antibody) with a dendritic marker (anti-MAP2 antibody). The result will be an overlap between the axonal marker and the dendritic marker which only be present in the AD brain slice.

Please, look through the references below for further reading.

Good luck with your experiments.

Aderemi.

Article Specificity of Anti-Tau Antibodies when Analyzing Mice Model...

Article A Oligomers Cause Localized Ca2+ Elevation, Missorting of En...

Hi Aderemi,

Thank you very much for sharing your knowledge and experience!

Sorry that I didn't make my question clear -- actually I am not studying anything related to AD model, but simply wanted to stain axons with Tau antibody on frozen tissue slices to study tissue response and remodeling after the implantation of neural probes in the WT brain. The brain region of the implanted probe is hippocampus, which is involved in AD; and also I saw people use Tau antibody to characterize AD progression. So it makes me concerned that whether using Tau is a wise choice in my experiment since it has some implications in neurodegenerative disease. If it is the case, then which subtype of Tau antibody I should use to have it decoupled from other implications? I raised this question because most of the papers I've seen use Tau for in vitro cell culture studies, but not much about tissue slices as I do, so I was wondering if there is a reason that people use neurofilament rather than Tau to stain axons on tissue slices?

Thanks a lot!

-Xiao

Hi Xiao,

Sorry for the delay.

You should use Tau as axonal marker, there are lots of commercially available anti-Tau antibodies (this is total Tau, not phosphoTau or any other post-translationally modified Tau). Since you are using mice, get a tau antibody that shows immunoreactivity against mouse tau, this is very important; you can easily confirm this from literature citations of that antibody. Also, I will like to add that Tau localization to the axonal compartment is an early embryological event, tau staining will always NORMALLY mark the axon.

As regards the neurofilaments, first you need to appreciate that there are light, medium, and heavy chain neurofilaments (NF-L, NF-M, NF-H; 70, 150, 200Kd respectively) and even though they are present in the axon, they are probably not exclusive to the axon. In addition, there levels in the axon is by far more than that of tau, so this could probably explain the reason why they are being used for staining in tissue slices which are understandably much thicker than cell culture for instance. So, because of the variation in the form of the NFs which might complicate your experiments, I will advice you to go with total tau as axonal marker. Whether you want to stain axon in cell culture or brain slices, tau is very reliable and consistent.

Regards,

Aderemi.

Thank you very much for your great advice, Aderemi! I really appreciate it.

-Xiao