n********k
发帖数: 2818 | 1 anyone has good experience with Tuj1 staining, it works for us but with
pretty background, would love to work out a better protocol for one study...
Any tips/informations would be highly appreciated |
R****e
发帖数: 53 | 2 how do you tell which is or which is not bg for Tuj1?
It is a very good ab. Try low conc. |
a**p
发帖数: 49 | 3 one way is to increase triton concentration
or inlcude if u hv not
it increase AB penetration and reduce background |
s******e
发帖数: 370 | 4 this shouldn't be very hard, tuj1 is one of the best antibodies
we order it from covance
also check other conditions as well, like 1st/2nd ab conc, species
reactivity, and so on. and have a positive control. |
n********k
发帖数: 2818 | 5 Thanks a lot, folks! Your responses light new hope in my despair heart:)))
Could I have your protocol and also a picture of your staining....
here is what we do: 30 um microtome brain section and focus on Hippocampus:
we mount the section and dry for several hrs and 0.01M(PH6) Citric acid
for 15 min at 95-100, and then
cool down twice in TBS for first time 2-3 min, second time 10 mins.
blocking with 3% DNS plus 0.3% Tween-20 for 1 hr and primary ON in
blocking solution,
(we tried anywh |
a**p
发帖数: 49 | 6 30 micron meter
well
u can do it in floating sections for staining
with triton rather than tween
staining overnight at 4 degree
TWEEN is too weak
if background too high
reduce concentration of 2nd Ab as well
not necessarily the 1st |
n********k
发帖数: 2818 | 7 Thanks again...tried free floating methods with virtually no staining at all in all
concentrations..I believe we tried Triton-X100
as well, but will check with my team, we also tried to even wash with 1-5% Triton-X100 afterwards from
anywhere 1 to ON...
BTW, we do lots of staining with many many different Abs but for for some reasons this has been one not
working well with the labs around here( maybe I am asking too much for this staining)...With that, I am really
looking for very specific/firs
【在 a**p 的大作中提到】
: 30 micron meter
: well
: u can do it in floating sections for staining
: with triton rather than tween
: staining overnight at 4 degree
: TWEEN is too weak
: if background too high
: reduce concentration of 2nd Ab as well
: not necessarily the 1st
|
s******e
发帖数: 370 | 8 tuj1 is a cytoskeleton protein, not sure if you really need the antigen
retrieval step (citric acid, boiling, etc), usually it's for nuclear
antigens
also are you doing adult or embryonic brains?
tuj1 level decreases in adult CNS but remains high in PNS, try a positive
control with embryonic brain sample |
