Help! Incredible high immunfluorescence background!

[Thorsten Schmidt]

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