Help! Incredible high immunfluorescence background!
Thorsten.Schmidt at rz.ruhr-uni-bochum.de
Tue Feb 24 13:45:26 EST 1998
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.
- different human cell lines: neuroepithelioma, neuroblastoma, glioma,
- 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 %
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
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
I would be happy about such "beautiful" experiments, but I would expect to
nothing because a anti-rabbit should not bind to any structures of my human
I made sure that there is no autofluorescence because an experiment without
secondary antibody resulted in no signals.
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.
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