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Demonstration of glycogen in tissue holds considerable diagnostic relevance across various pathological conditions, particularly in certain tumors. The histochemical staining of glycogen using methods utilizing Schiff’s reagents is subject to influences arising from the type of fixative, fixation temperature, and oxidizing agents employed. This study aimed to assess diverse fixatives, fixation temperatures, and oxidizing agents, each with variable treatment durations, in conjunction with Schiff’s reagent for optimal glycogen demonstration. Paraffin blocks derived from a rabbit’s liver served as the experimental substrate, encompassing 340 paraffin sections subjected to different procedures. For tissues fixed at 4 °C, good staining outcomes, as determined by the periodic acid–Schiff (PAS) stain, were observed with 10% neutral buffered formalin (NBF), 80% alcohol, and Bouin’s solution. Tissues fixed at room temperature (RT) demonstrated good PAS staining results with both 10% NBF and 80% alcohol. Notably, other oxidizing agents exhibited poor outcomes across all fixatives and fixation temperature, with two exceptions, as satisfactory staining results were obtained when using 5% chromic acid. Consequently, Both 10% NBF and 80% emerge as preferred fixatives of choice for glycogen demonstration when coupled with PAS stain. It is noteworthy that Bouin’s solution could also provide good outcomes when fixation occurred at 4 °C.

Demonstration of glycogen can be done in different lesions and is considered diagnostically significant, mainly in some tumors. Glycogen staining is affected by the type of fixative, the temperature of fixation, and the staining technique.Grocott’s methenamine (hexamine) silver technique quality was assessed after four different types of fixatives at two different temperatures [Bouin’s solution, 10% neutral buffered formalin (NBF), 80% alcohol, and Rossman’s solution at room temperature (RT) and 4 °C, for 24 h]. These variables were studied to optimize this technique for glycogen demonstration. Archived paraffin blocks were used in this study. They were prepared from one rabbit’s liver, and 32 paraffin sections were prepared and stained with Grocott’s methenamine (hexamine) silver technique. Eighty percent alcohol provided the highest staining quality scores at both RT and 4 °C in comparison with the other fixatives. We concluded that 80% alcohol at 4 °C seems to be the fixative of choice for glycogen with the Grocott’s methenamine (hexamine) silver technique at the level of this study.