[Histonet] Glycogen detection; also Spring Forward

[John Kiernan]

Glycogen (MW about 1,000,000) is soluble in water but insoluble in alcohol (Merck Index 12th ed.,1996, p.766). For this reason, non-aqueous coagulant fixatives may have advantages, especially for small specimens or thin layers of cultured cells.

Fixation immobilizes cytoplasmic proteins, which then entangle the big long polysacccharide molecules of glycogen, keeping them  approximately in their intracellular positions. Formaldehyde penetrates specimens rapidly, but its chemical reactions with proteins, especially the cross-linking that stabilizes structure, are slow (meaning 12-48 hours). During this time, the glycogen in liver cells dissolves and is carried in solution along the direction of  the fixative diffusing into the specimen. In each hepatocyte, this intracellular diffusion of glygogen is stopped by each hepatocyte's cell membrane, which has a lipid layers that are unchanged by an aqueous formalin solution. As a result, the stainable glygogen piles up in the side of each hepatocyte furthest from the surface of the specimen. This artifact is often called "polarix=zarion". With processing into paraffin, which removes lipids and coagulates any proteins not yet made insoluble by formaldehyde, glycogen is anchored into place by fixed cytoplasmic proteins, but it can still be attacked and removed by amylase/diastase/spittle.

All this has been known for at least 60 years. It's in the textbooks, as Bob Richmond pointed out yesterday. (Or was it the day before?)  It's now time for us all to advance our clocks by an hour, go to bed and wake up in time for Church on Sunday!

John Kiernan
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________________________________
From: Bob Richmond via Histonet <histonet at lists.utsouthwestern.edu>
Sent: 07 March 2020 13:47
To: Histonet at lists.utsouthwestern.edu <histonet at lists.utsouthwestern.edu>
Subject: Re: [Histonet] Glycogen detection

Galina Deyneko (where? asks: >>Does anybody have experience how fix the
tissues for successful glycogen ? detection in murine and humane
cardiomyocytes. I am wondering maybe the trace of methanol in 10% formalin
will dissolve glycogen?? - What would be better process for paraffin
embedding or use OCT embedding without fixation? Of course I prefer FFPE
blocks, since OCT blocks give bad morphology.<<

Ordinary neutral buffered formalin and paraffin embedding should be
adequate. R.D. Lillie (3rd ed.) notes good results with Carnoy's fixative,
alcoholic formalin, and acetic alcoholic formalin also.

The traditional stain for glycogen is periodic acid Schiff (PAS). You
verify the presence of glycogen by doing the stain with and without amylase
("diastase") predigestion. (A crude but adequate source of amylase is to
just spit on the slide.)

Bob Richmond
Samurai Pathologist
Maryville, Tennessee
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