Comparison of Three Staining Methods of Vibratome and Paraffin Sections in Preserving Thickness of Rat Hippocampal Sections

Purpose: To determine the optimal technique for maintaining hippocampus vibratome sections volume. Materials and Methods: This experimental study was performed on three rat hippocampus. The first hippocampus was embedded in paraffin and using rotary microtome forty sections were cut in a nominal thickness of 100 micrometer. They were then ordinary stained. The two others were cut into 80 sections by a calibrated vibratome to 100μm thick sections. The first 40 series of vibratome sections were routinely stained and the second sections stained free-floating and embedded in resin with thionin. Section thickness was measured with a microcator mounted to a microscope. The distribution of neurons in section’s density was assessed. Results: The findings showed that Means±SD in free-floated and routine-stained vibratome sections were 74.5±6.18, 41.2±4.33 respectively while in paraffin sections was 46.3±5.95. Also the results indicated that neuronal density in the upper and lower surface of free-floated sections was lower while the distribution in the 70% of the middle part of the section was fairly uniform. Neuronal density in the upper and lower surface of ordinary stained vibratome sections was higher. Paraffin sections had higher neuronal densities at upper and lower surface while a lower and nonuniform distribution of neurons was observed at the central layer. Conclusion: The most critical stage in routine staining procedure of vibratome sections is the duration that they are dried in air and on glass slides that results to permanent reduced thickness of sections. However in resin embedding procedure the reduced thickness of sections was minimal because the sections are not exposed to air at any stages. On the other hand, the consistent distribution of neurons in the most part of section thickness allows one to estimate sterologically the number of neurons in hippocampus using vibratome sections.