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sections of tissue (specifically goldfish neural retina) fixed with 4%

paraformaldehyde? We are interested in the light level, not EM.

Linda Barthel

Research Associate II

Department of Anatomy and Cell Biology

University of Michigan

lab (313) 764-7476

fax (313) 763-1166

barthel@umich.edu

Periodic Acid Schiff is first favourite. Any histochemistry

text will give you the necessary details, failing that, e-mail me and I can

send you my protocol.

Ian.

I.Montgomery@bio.gla.ac.uk

sent her some background information needed in the microscopic study of

glycogen, and I repeat them for all researchers interested in the subject.

The problem of glycogen is more complex than commonly appreciated, and the

understanding of this complexity is a sine qua non condition in the

microscopic study of glycogen.

Glycogen in the cell appears in the organelles, GLYCOSOMES, composed of

glycogen and enzymes involved in glycogen synthesis and degradation. The

structures stained by uranium and lead salts, and interpreted in EM as

"particles of glycogen" represent in fact the protein component of

glycosomes. Glycogen does not react with the ionic stains, but it can be

demonstrated histochemically, by the PAS technique (periodic acid - Schiff

reagent) in LM, and by the modification of this procedure (Thiery

technique) in EM.

The problem is that glycosomes are easily destroyed during tissue

processing. The most common destructive factor is the change in pH which

breaks the bond between glycogen and protein. The effect is that the

soluble protein component (enzymes) is washed out, and glycogen which is

not fixed, floats in the cell and aggregates into clumps. The acidic

treatment is inherent in the PAS procedure where periodic acid is used,

therefore, in the vertically processed slides, the unfixed glycogen often

accumulates as crescents at the bottom of the cells (the effect well known

in the classical histochemistry).

In EM the common destructive factor is uranyl acetate (strongly acidic)

used before tissue dehydration. In tissue processed without uranyl acetate

glycosomes appear intact, even after the priodic acid treatment in Thiery

technique, because the histochemical reaction is performed on sections

which are already embedded in the resin. This embedding prevents the

floating of the unfixed glycogen.

Freezing seems to be another destructive factor for glycosomes. Raether et

al, 1977 (Z.Parasitenkunde, 54, 149) used deep-freezing of Entamoeba

cultures, and found that only a few amoebae retained normal structure.

Their micrographs indicate that in the destroyed organisms the glycosomes

were destroyed.

Additional complication is that the described factors affect only free

glycosomes, whereas others, which are bound to different cell structures

remain resistant.

The review of glycosomes was published by

K.K.Rybicka, 1996, Tissue & Cell 28 (3) 253-267.

Best wishes in further study,

Krystyna

rybicka@acsu.buffalo.edu