geez. i have 2 suggestions.
rabbit IgG are notorious for being VERY sticky and non-specific. that
is because rabbits scratch at themselves in their cages and make alot
of autoantibodies and antibodies against all kind of stuff floating
around in the air. you must therefore switch to guinea pig or mouse
IgG OR affinity purify your antibody.
since you listed alot of info, you have reduced it to your rabbit IgG.
and you got it from Boehringer. have you called them?
cells in culture suck up serum proteins, and your antigen may be present
in your bovine serum used to grow the cells.
Jim Bassuk
U Washington
Seattle
On 23 Feb 1998, Thorsten Schmidt wrote:
> Dear reader!
>> I have biggest problems with immunfluorescence of cover slips.
>> I would like to make immunfluorecence experiments with cell culture cells
> grown on cover slips.
> But I get strong signal even with only the secondary antibody.
>>> I tried
>> - different human cell lines: neuroepithelioma, neuroblastoma, glioma,
> astrocytoma
>> - different fixation techniques: Methanol (15 min. -20°C);
> Methanol:Aceton 1:1 (15 min., -20°); 3,7 % Formaldehyde (10 min.)+1 %
> Triton (10 min.)
>> - different blockage methods: 1 % BSA in PBS; 100 % fetal calf serum; 5 %
> normal goat
> serum + 0,2 % Triton
>> - preblockage of aldehyde groups with tris-glycin
>> - different dilutions of secondary antibody.
> (boeringer mannheim anti-rabbit-FITC F(ab)2-fragment)
> (boeringer recommends a dilution of 1:4 to 1:10 and I tried dilutions up to
> 1:20).
>> but had always the same problem:
>> INCREDIBLE HIGH BACKGROUND!!!
>> WITHOUT any primary antibody I get "very nice" results:
> Staining of cells, nuclei, cytoplasm etc. but not of the intercellular
> area.
> I would be happy about such "beautiful" experiments, but I would expect to
> see
> nothing because a anti-rabbit should not bind to any structures of my human
> cells!
>>> I made sure that there is no autofluorescence because an experiment without
> secondary antibody resulted in no signals.
>> Help!
>> What else can I do? I am completely helpless!
>> How can I reduce the background?
>> Can you recommend a different antibody or coupled dye which works better?
> Perhaps a "Cy"-dye?
>> Or do you have any experiences with this antibody
> (boehringer mannheim anti-rabbit-FITC F(ab)2-fragment)?
>> Could you please mail me a working protocol for immunfluorescence?
>> Or do you have any other ideas?
>>> I am deeply grateful for every hint.
>> Thank you so much in advance.
>> Best regards
>> Thorsten Schmidt
>>>