Comparison of formaldehyde-preperfused frozen and freshly frozen tissue preparation for the in situ hybridization for alpha-tubulin messenger RNA in the rat brain

In situ hybridization histochemistry, using the alpha-tubulin oligonucleotide probe, was performed employing two tissue freezing methods, the preperfuse-freezing method and the freshly frozen method, to evaluate methodological differences in the messenger RNA (mRNA) levels of the brain. The mRNA levels in the sections from the freshly frozen groups were lower than those in the preperfused group, particularly in the cerebral cortex, the striatum, the hippocampal CA1 and CA4 fields, and the habenular nuclei. Therefore, employing the freshly frozen method, there is a possibility that the mRNA levels in these regions may be underestimated. The hybridization signals of groups placed on an ice-bed for 5 min and 15 min were lower than those of the immediately frozen group in the cingulate, the temporal, and the retrosplenial cortex, the CA1 field, and even in the medial and the lateral thalamic nuclei where no significant difference was seen between the perfused group and the immediately frozen group of tissues. When employing the freshly frozen method, the removed brain should be frozen as fast as possible and the period from decapitation to freezing should be kept strictly constant.