Help! Incredible high immunfluorescence background!

Marieke R. Koedood Zhao rkoedood at
Thu Feb 26 14:46:09 EST 1998

I have very little experience but here is my 2c. worth...

The general opinion here from the guys with a lot of experience is that 
too much Ab will give you high background. Our FITC-Ab are not from
Boehringer, so what I say may be irrelevant. However, for all secondaries
that I've used thus far, I used dilutions ranging from 1:50 to 1:200.

We buy Ab now from the Jackson labs - they have some that have been 
pre-absorbed to avoid species-cross-contamination. If you don't get out
of your background-hell by diluting the Abs, maybe you should try getting
them from Jackson (Tel. (800) 367-5296 or (610) 869-402, no affiliation, just
happy with their product)

Good luck


Thorsten Schmidt (Thorsten.Schmidt at 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:


: 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

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