The histological processing of teeth is highly complicated because of containing both mineralized hard tissues and soft tissues. Depending on the type of decalcification agents used in processing, mild-to-severe deterioration in the tissue structure and inadequacies on clear staining of details by the histological stain may be observed. This study aims to compare the histological staining differences in the preparations from decalcified and undecalcified tooth roots by three different embedding materials and techniques. Following extraction, human single-rooted teeth crowns were cut off and roots were placed in 10% buffered neutral formalin. After fixation, roots were divided into two groups. One part of samples was decalcified in formic acid solution and the other was remained undecalcified. Decalcified roots were embedded in paraffin and glycol methacrylate (GMA)-based resin and undecalcified roots were embedded in methyl methacrylate (MMA)-based resin. Sections from all groups were stained with hematoxylin and eosin. The groups were compared in terms of general staining, brightness, density, density of the base stain, general morphology of cells, nuclear/cytoplasmic contrast, distinguishability of pulp, odontoblast layer, predentin and dentin, preservation and traceability of dentinal tubule. In the preparations which were embedded into the MMA-based embedding material, an output lower than the paraffin group but higher than the GMA-embedded group was provided. As a result, the best histological detail was obtained from the decalcified, paraffin-embedded sections.