Autofluorescence elimination from cell aggretates - Immunostaining troubleshoot: Autofluorescence (Nov/07/2005 )

Dear people,

I am doing immunostaining on aggregates of cells and realize that there is quite a considerable presecence of autofluorescence of red or green (co-localized) when I switch the viewing channel of my fluorescent microscope. I am using Zeiss Upright AxioImager FL microscope equiped with band pass filters which should eliminate bleed-through. Even more unusual is the presence of green when only Cy3 secondary Ab was used!

I am using 4% paraformaldehyde/1hr for fixing the cells aggregates formed under "hanging drop" and attached to coverslips for 4hrs.

I permeabilized with 0.3% triton-X (PBS)/30mins, block with 10% goat serum/30mins and overnight incubation with 1.Ab at 4C. Secondary Ab (Cy3) at 37C/30mins. 3x wash with 0.3% trinton-x (PBS)/5mins each. DAPI and mount the coverslip on glass slides.

Alternatively I used FITC before in the place of Cy3 and notice red autofluorescence! I am thinking of using the avidin/biotin block but would like to know if it is relevant to the elimination of autofluorescence be it red or green.

The autofluorescence is quite obvious when the attachment of the aggregates is over 3 days as opposed to 4hrs.

Anyone could enlighten me on autofluorescence troubleshooting. Help is much needed and deeply appreciated.

i don't know if this is the same problem i have but i get positive fluorescing cells in my negative controls [no primary antibody] and one of my colleagues gets positive cells when they haven't even had the secondary-FITC conjugated antiocody added to them!