Abstract
The rates of enzymatic glycogen removal from two different types of rat liver preparations were determined histochemically, employing the periodic acid-Schiff method to visualize glycogen. The first preparation consisted of elongated liver blocks (approximately 2 x 2 x 10 mm) which were fixed, then exposed to the alpha amylases before dehydration and embedding. The second type of preparation consisted of 5-8 µ thick, routinely prepared sections. The glycogen was removed considerably slower from the formaldehyde fixed tissue blocks than from the sections. The rate of glycogen hydrolysis was enchanced in the liver blocks by brief periods of autolysis, by removal of lipids and by washing before exposure to the enzyme.
Hydrolysis by amylase was slower when 0.18 M phosphate was used with the formaldehyde during fixation than when veronal acetate or 0.018 M phosphate buffers were used at the same pH 7.2-7.4. Many peripherally located cells retained glycogen even after incubation of the entire blocks for 24 hours in alpha amylase when the buffer during fixation was 0.18 M sodium Phosphate.
The contaminating ribonuclease present in malt diastase preparations penetrated readily into the cells of the elongated liver blocks but the protease affected only the periphery during the 24 hour incubation.
The differences in resistance to enzymatic hydrolysis were not confined to the tissue blocks. The type of buffer and fixative used for tissue fixation determined the subsequent susceptibility of glycogen to hydrolysis in sections also. The list is arranged in order of progressively decreasing resistance to enzymatic hydrolysis: 3.8% formaldehyde or 3% glutaraldehyde with 0.18 M phosphate, 3.8% formaldehyde with 0.018 M phosphate or 0.18 M veronal acetate, 3% glutaraldehyde with distilled water, 3.8% formaldehyde with 0.129 M veronal acetate or distilled water, and 70-95% ethanol. In addition, glycogen was removed slower from the peripheral zones of the sections than from the central portions.