4 replies to this topic

[thumbclaw]

Hi all,

I have been working on PAS stain with cultured cells, but I supposed this subforum would have the most experienced persons.

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.

[mdfenko]

after digesting with amylase, the remaining staining is of glycogen (and glycoproteins and proteoglycans). if there is no change then there was no stained starch.

if you digest with diastase then you will reduce the staining of glycogen.

[thumbclaw]

mdfenko, on 28 October 2010 - 08:58 AM, said:

after digesting with amylase, the remaining staining is of glycogen (and glycoproteins and proteoglycans). if there is no change then there was no stained starch.

if you digest with diastase then you will reduce the staining of glycogen.

You just blew my mind.  Now I am thoroughly confused.  <Searches through references scattered all over desk>

[thumbclaw]

thumbclaw, on 28 October 2010 - 09:04 AM, said:

mdfenko, on 28 October 2010 - 08:58 AM, said:

after digesting with amylase, the remaining staining is of glycogen (and glycoproteins and proteoglycans). if there is no change then there was no stained starch.

if you digest with diastase then you will reduce the staining of glycogen.

You just blew my mind.  Now I am thoroughly confused.  <Searches through references scattered all over desk>

I have found several sources saying that alpha-amylase depolymerizes glycogen into smaller sugars which get washed away.  Can you direct me towards your source please?  Many many thanks!

[mdfenko]

according to the worthington enzyme manual (and catalog, with references) alpha-amylase acts on long linear polymers.

that statement i made about diastase was ignorant and i apologize. i was looking at pas staining and the source said that diastase would digest the glycogen. i have since been educated that diastase is amylase (not specific to alpha). so i'm not sure that it will digest glycogen.