RNA in situ hybridization: pH of AP staining buffer? - (Jul/16/2012 )

Hi,

I'm starting to do RNA in situ experiment and I have a working protocol for that. I just wonder the recipe of the AP staining buffer:

100 mM NaCl
50 mM MgCl2
100 mM Tris pH 9,5
0,1 % Tween-20

Why the pH is that high? I have checked also other protocols as well as kit manuals and they say the same. Tris isn't working as a buffer at pH 9,5!

9.5 is just outside the buffering range of tris. it will resist change of pH towards the acidic.