PAS is a broadly used staining method to quantify glycogen content in muscle. As you suspect, it also stains mucin and basement membranes, but in any case the contribution of these components, in terms of quantification of glycogen depots within skeletal muscle fibers, should be very modest. In my experience, I can tell you that it's rather easy to identify glycolytic and oxidative fibers with this staining, based on staining intensity. I'm not very confident that it is that much sensitive to allow discrimination of small differences. Anyway, it is very simple to perform, so it's worthy to give it a try.

Actually, i work with glycated proteins too. I fear if they are stained with PAS too, ( pas has been used to stain glycated proteins too). How will I be able to differentiate them in the cell then?

Not,  the  PAS staining  is  not  specific  for  glycogen, while  for aldohexoses. All material which  contain  aldohexose (glucose, mannose  etc.) is  PAS  positiv. In  contrary,  the  ketohexoses (for  example. the  fructose) is  PAS  negative.The  glycoproteins   contain  aldohexoses  also, while  numerous  other  substantia   contain aldohexoses, for  example  dextran,  starch, pectin,. The  differentiation  between  glycoproteins  and  glycogen  is  simple. If  you exam,ine  parallel  slides, one  with  "simple"  PAS  staining, and  another  after  diastase  digestion. If  the PAS  staining  become  negative  after  digestion,  than  this  material  was  glycogen.

The  color  intensity  of  PAS  staining  is  not  stochiometric  with  the  glycogen .

Józsa L.: Untersuchung über den Zusammenhang zwischen Menge,

Polymerisation unf Farbenintensität der Polysaccharide.

Acta Histochemica, 32, 305-310, (1969)

.

Józsa L., Perneczky M., Lusztig G.: Histologische Beobachtungen mit Dextran und

bei experimenteller Cholesterinsklerose.

Zentralbl. Allg. Pathol. 103, 200-210 (1961)

With best regards: LG.Józsa

I believe that diastase digestion will act as a negative control and removes glycogen.  If you digest sections with diastase (37 °C for 1 hour) before PAS staining, it should be negative for glycogen.  Protocols are available online.

@Prof. Jósza László: thank you really for the interesting and acknowledged answer. Would it be possible to get either a copy of your papers or eventually also a scanned pdf of these articles? Thank you for your kind reply, regards, Wolfgang

@Meha Fatima:

perhaps you have found already an older, but for the matter you are to be examining  also interesting paper, published by  Gert Schaart et al.:

A modified PAS stain combined with immunofluorescence for quantitative analyses of glycogen in muscle sections, in:

Histochem Cell Biol (2004) 122:161–169    DOI 10.1007/s00418-004-0690-0, via PubMed see  http://www.ncbi.nlm.nih.gov/pubmed/15322861, the pdf -if you can access for free available at http://link.springer.com/article/10.1007%2Fs00418-004-0690-0. (if you cannot access for free, write to me and I will be able to send you a saved pdf). Following such a technique it might be able to solve your problem.

Best wishes and regards

Wolfgang

PAS stain not only stain glycogen but it's also stain mucopolysaccharide, parasite such as ameoba and fungi.

@Wolfgang H. Muss, thank you for posting that article, and many thanks to Prof. Józsa for the precious informations that you provided.

@Prof Józsa: in the light of what you wrote previously, may I ask you to comment on the quantification method proposed in the paper posted by WH Muss? They draw a correlation between quantification of glycogen content biochemically measured in muscle extracts and glycogen measured on images after PAS staining. I guess this is possible only if the great majority of the PAS staining signal comes from glycogen. But if the relationship between colour intensity and glycogen is not stoichiometric, as you said previously, how accurate can be the method proposed?

Best wishes

Stefano

Dear Stefano Ciciliot

& Prof. Józsa,

both your comments have been extremely valuable also for me (having dealt with a lot of PAS as well as "PAS-Like" stainings (latter on semithin resin sections).

Stefano, you're welcome with your reply as well as further questions. For me arises another question too: namely, the relationship between "primary FIXATION" & temperature (since there one for sure can expect a certain percentage of "eluted glycogen stuff" during and post processing anyway), processing and after embedding into paraffin (cave also : section thickness etc., optic system of analysis) STAININGs  is/are not only PAS and perhaps some variants for localizing Glycogen in tissue sections. For this reason I would like only to cite the following 2010 paper out of my files:

by Yosef Mohamed Azzam Zakout , Magdi M Salih, HG Ahmed in: Biotechnic & Histochemistry, 2010, 85(2): 93–98                                                                                (keywords: Best's carmine , fixative, glycogen , hexamine silver, PAS, temperature).

Besides reading the original article (unfortunately only access via PPV online or via library lending if not available in your library, i have a pdf in my files) perhaps would be a good option, they state in their :

"For the PAS reaction, Bouin's fixative gave better results at both temperatures compared to the other fixatives. For hexamine (methenamine) silver, the quality of staining was improved for tissues fixed in both 10% formalin and NBF at 37°C compared to Bouin's solution. Both 10% formalin and NBF at 4°C gave better results than Bouin's solution. For Best's carmine, Bouin's solution gave the best results for tissues fi xed at 4°C. Fixation of tissues with NBF at 37°C gave the best quality staining. We concluded that the quality of glycogen staining in paraffin sections is greatly affected by both the fixative and the temperature of fixation."

Amazing results (but "nothing is impossible")!

Bottom line: One never should stop "digging" in recent and older literature...

Very best regards and luck to all, especially also to Meha Fatima, Wolfgang

Thank you so much all the intellectuals! @ wolfgang. Thank u so much. I'll be grateful if you can send me the paper at [email protected]. thank you so much once again.

PAS with and without digestion is specific for glycogen. This technique works by the enzyme extracting only the glycogen within the tissues. You can use diastase, alpha amylase or saliva to depolymerize glycogen and break it into smaller sugar units that wash away. You need to run parallel slides simultaneously during digestion. (2 slides sectioned from the same block. 1 slide goes into the enzyme digestion and the other slide does not, it is left in water or buffer. Then stain the digested and undigested slides together with PAS and compare results.

Best's Carmine method is better for glycogen as PAS stains up so may other components too, but you must use an amylase control.  I have also found that the lectin BSA-II stains glycogen very strongly, though I am not sure whether oligomers also come up too. The binding is certainly removed by amylase action.

What if I stain 'cell line' or 'cells' with PAS only? Is there any intracellular protein that can be stained with PAS? If not, then I might be able to use simple PAS stain only, right?

There are quite a few carbohydrates that stain with PAS and it is certainly not specific for glycogen. You can control the staining with amylase digestion i.e. glycogen staining will disappear but the rest will be left.

Hi Meha

PAS  is used alone for detect carbohydrates, polysaccharides, microvilli, and basement membranes ; PAS- Alcian blue used for detect mucopolysaccharides , mucin and acid mucin. 

Hello all,

I'm also trying to do similar experiment. Although I'm trying to stain cells in 24wp format which are fixed with 4% paraformaldehyde. So they are a bit sensitive. Cells start to shrink and some of them even come off the well when I do 0.1% diastase treatment prior to PAS staining. 

Has anyone tried staining cells in such format instead of tissue sections? Any help would be much appreciated.

Thanks,

Kalpesh

Yes Kaplesh. Try fixing with methanol. Even I got my cells detached after fixing with 4% PFA. I did not treat them with diastase but cells will remain attached I believe because methanol did it quite well. Also see this for detailed method:

Aftab MF, Afridi SK, Ghaffar S, Murtaza M, Khan M, Karim A, Khan KM, Waraich RS. A bis-Schiff base of isatin improves methylglyoxal mediated insulin resistance in skeletal muscle cells. Archives of pharmacal research 2015.

Thanks Meha. I will give it a try.