PAS negative - amylase or diastase digestion (Oct/28/2010 )

Hi all,

I have been working on PAS stain with cultured cells.

I have read in many papers that PAS stains were "subsequently digested with Amylase" to show true glycogen stain, but have found neither pictures nor protocol to do this myself.

I have been using a-amylase from porcine pancreas (sigma A3176-1MU) diluted in water (although it does not fully dilute and sigma says it should and sent me another bottle which still does not) at 1%-5%, and have incubated at 37deg anywhere from 30 minutes to overnight with absolutely no change in the PAS positivity.

I have also tried incubating with similar solution prior to PAS stain, but still my cells are fully magenta-- if I fix. If I don't fix they just come right up with the amylase digestion-- unless the cells are on collagen- but even then many of the cells come up-- and those that don't are stil staining magenta.

I would really love some help with this. I can't figure out what I am doing wrong

I don't know the answer to your questions sorry, but you could try the forums at IHC world who specialise in this sort of thing.