Multiple Immunofluorescence Labelling in Frozen Sections

From:Kelvin So

Hello Histonetters,

I am working on a protocol for multiple immunofluorescence labelling on
frozen sections using Alexa fluors 488, 546 and 647 and I need some
information for the following questions:

1) Will the addition of Tween-20 to the washing PBS have any damaging
effect on the fluorochromes in terms of specific binding to the markers
concerned as well as on the subsequent stability of the fluorescence in
the final preparation?
2) My markers of interest are anti-Thioredoxin, anti-APE/Ref-1 and
anti-CA IX.
Is paraformaldehyde/formaldehyde the best choice of fixative for these

Any feedback on these questions or any well-established protocol on
multiple labelling techniques will be appreciated.

Kelvin So
University Health Network
Toronto, Ontario.

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