Aldehyde Autofluorescence - Fluorescing controls a problem (Jul/29/2005 )
Hi, I'm relatively new to tissue culture and immunochemistry so when I read a post that mentioned that aldehydes autofluoresce, I was intrigued. I've been having problems with my controls fluorescing when I haven't added any Rhodamine conjugated Ab to them. I am fixing all my samples (including controls) with 4% formaldehyde and wonder if maybe that is my problem. I do wash my cells with PBS after fixation, but perhaps I am not washing them enough. Any insite would be helpful!
This is a famous issue when using PFA for fixation of cells and tissues.
You can easily quench aldehyde groups by incubating in glycine 100mM in PBS for 20 min.
Other methods include sodium borohydride, sudan black, detergent solubilization etc etc.
when i use PFA for fixing cells after staining with fluorochrome-coupled Abs, i only observe a slight shift in the fluorescence intensity by flow cytometry and washing with PBS after fixing don't decrease this effect
on the other hand autofluorescence is really important when using methanol in immunocytochemistry...
why such a difference?