We are doing intranuclear staining for transcription factors, which requires a methanol fixation/permeabilization step. We are finding that the methanol fixation dramatically reduces APC fluorescence (of cell pre-stained with mAb). Staining protocol (simplified) 1. 4% PFA fix 2. stain for surface markers (FITC, PE, APC conjugated) 3. 80% methanol (-70 C) fixation 4. Perm with 0.5% saponin, stain for transcription factors with PE/Cy5 labeled secondary mAbs 5. Run We find that methanol fixation reduces or eliminates the APC fluorescence, but does not change the PE. Questions: 1. Has anyone else seen this? 2. Why doesn't it change the PE fluorescence? 3. Solutions? The only thing I can think of is to switch to Cy5.
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