Hi Pranay,

I'm not aware of all the conditions you work with (like Rodolphe suggests), but if you see very faint staining, and if you can confirm you have no staining what so ever on a negative control, I would suggest you just have to amplify the signal (probably because tau expression is low?). For example, I guess you secondary antibody is labeled with a fluorophore, but you can upgrade detection by incubation with a secondary antibody labeled with biotin, after which streptavidin-HRP can bind, allowing visualisation via the TSA-Cy3 system. Or, you can just dilute your primary antibody 1:100 to start with. It could also be very interesting to take with a positive control, where you are certain to find strong tau expression, so you can confirm the antibody is specific.

Best of luck

Hi Pranay,

I'm not aware of all the conditions you work with (like Rodolphe suggests), but if you see very faint staining, and if you can confirm you have no staining what so ever on a negative control, I would suggest you just have to amplify the signal (probably because tau expression is low?). For example, I guess you secondary antibody is labeled with a fluorophore, but you can upgrade detection by incubation with a secondary antibody labeled with biotin, after which streptavidin-HRP can bind, allowing visualisation via the TSA-Cy3 system. Or, you can just dilute your primary antibody 1:100 to start with. It could also be very interesting to take with a positive control, where you are certain to find strong tau expression, so you can confirm the antibody is specific.

Best of luck

Thanks Pieter. I will try both the ideas. I did a positive control but the staining was still faint, but when I stained the control neurons (with no construct in it, hence no GFP) with secondary antibody labelled with green flourophore a saw a little better staining than with red flourophore. But I need the red flourophore to work.

Hi Rodolphe Hajj, No I don't want to keep the cells living, I fix it before staining, I just need the staining to work properly. Though it looks like a pretty straight forward staining, I don't know why but it's just not working.

Thanks Rodolphe for the suggestions. I have never tried any dilution below 500, also I have not tried any other tau antibody. Yes I do permeabilize the cells as well.

Dear Pranay,

I don't know about the anti- tau antibody you purchased, but we have had good luck with the clone Tau-1 (PC1C6).  We originally purchased it from Chemicon, but it is now available from Millipore:

http://www.emdmillipore.com/US/en/product/Anti-Tau-1-Antibody%2C-clone-PC1C6,MM_NF-MAB3420

When using only a labeled secondary, we had to use at least 5ug/mL, which for their packaging was a 1:200 dilution.  To truly be able to see detect it in very thin axons in vitro, we had to use a 1:75 dilution with the labeled secondary.

In addition to the suggestions of Rodolphe and Pieter, you might also try labeling with the primary antibody overnight at 4 C, rather than 2 hours ar room temp.

How are you detecting the red fluorophore, and which one is it?  Certain ones are difficult for the human eye to detect, but CCD cameras for the microscope are much better at it. 

Good Luck!

Jill

Remove the tagging and they will all be showing bright red with staining method.

Thanks Jill. So it looks like that you use a much lower dilution of secondary than what I use. So I will take your suggestion and use a lower dilution. Also overnight at 4 degree sounds convincing, I will try that as well. Thanks a lot.

For detection we have CCD cameras only.

Sorry Jai sir I didn't understand what tag are you talking about?

Dear Pranay,

I see now that what I wrote was unclear. I was only making suggestions for the dilutions of the primary mouse anti-Tau-1.  I meant that when we detect the primary antibody using a labeled secondary (as opposed to ABC amplification or a labeled streptavidin), we dilute the PRIMARY antibody to 5 ug/ml.  At that time from that company, that was equivalent to a 1:200 dilution.  When we were looking at regenerating adult mouse retinal ganglion cell axons in vitro, which are very thin, we had to dilute the PRIMARY to 1:75.

In order to detect this primary antibody, we were using a FITC-labeled goat anti-mouse IgG (H+L).  Newer fluorochromes can be much brighter, so you should use whatever dilution of your secondary antibody that has worked in the past in your system.

Sorry for the confusion!

Good Luck-

Jill    

No problem Jill Thanks a lot. I am working on it.