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
= = =
________________________________
From: Bob Richmond via Histonet <histonet@lists.utsouthwestern.edu>
Sent: 07 March 2020 13:47
To: Histonet@lists.utsouthwestern.edu <histonet@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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