Bright dots in immunocytochemistry - no real signal (Aug/09/2007 )
HI there,
I was trying to visualize some receptor on newborn DRG cells in 8 well chamber slide by targeting it with APC-labeled ligand, then goat anti-APC, then chicken anti-goat IgG labeled with texas red. The negative control is no APC-labeled ligand. The coating material is poly-L-lysine and laminin
goat anti-APC is 2 yrs old stored at 4 degree, from Biorad
chicken anti-goat IgG is 1.5 yrs old at 4 degree, from Invitrogen
What I observe is no staining signal, instead there are quite a few bright dots with unknown reason scattered around in the 8 well chamber slide.
Can anybody suggest what are the bright dots? why are they there and why no staining at all other than the bright dots? Thanks!
I was trying to visualize some receptor on newborn DRG cells in 8 well chamber slide by targeting it with APC-labeled ligand, then goat anti-APC, then chicken anti-goat IgG labeled with texas red. The negative control is no APC-labeled ligand. The coating material is poly-L-lysine and laminin
goat anti-APC is 2 yrs old stored at 4 degree, from Biorad
chicken anti-goat IgG is 1.5 yrs old at 4 degree, from Invitrogen
What I observe is no staining signal, instead there are quite a few bright dots with unknown reason scattered around in the 8 well chamber slide.
Can anybody suggest what are the bright dots? why are they there and why no staining at all other than the bright dots? Thanks!
Ab titer should be reduced to optimize visualization of structures; may be there are aggregates of your protein which accumulate the Ab;
are the dots disseminated or perinuclear or adjacent the plasma membrane or other remarkables?
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Ab titer should be reduced to optimize visualization of structures; may be there are aggregates of your protein which accumulate the Ab;
are the dots disseminated or perinuclear or adjacent the plasma membrane or other remarkables?
[/quote]
The dots are disseminated, and not prenuclear or adjacent the plasma membrane, the sites are rather random. 3 or 4 together here and there...I am thinking whether they are the result of interaction with coating material by the reagent, due to...?
Thanks!
Thanks!
If the dots are due to coating material, then you would see them in the negative controls also. How does it look in the control without primary antibody and secondary only. Could it be due to secondary binding alone or ineffective washing.
Stupid Question: did you use NGS for blocking? (I did this once, and then used a anti-goat 2ndary antibody...interesting result 
If that's not the problem, I'd try another secondary antibody. And if that doesn't work, I'd order a new primary antibody, because 2 years in the fridge don't really increase the efficiancy...
Good Luck!