Preparation of high-quality successive paraffin sections of rat brain tissue / 第二军医大学学报

ABSTRACT

Objective To investigate the methods of preparing high-quality successive paraffin sections of rat brain tissue. Methods Rats were anesthetized and transcardiac perfusion fixation was performed for collecting brain tissue. Then the brain was sequentially cut into 3mm-thick blocks and immersed in fixative; dehydrated in a gradient ethanol series, with 95% ethanol 2 times for 1 hour each, and ethanol 2 times for 30 minutes each; cleared with xylene 2 times for 10 minutes each; dipped wax at 60° with paraffin of melt point 56~58° for 1 hour then followed by melt point 58~60° for 2 hours; and embedded with the same paraffin as the second waxdip. 4μm sections were sliced with new knives, flattened with 42° water bath, and attached with adhesion slides. Results After perfusion fixation the brain tissue appeared milk-white and had certain toughness; through xylene clearing the brain presented totally transparent, without any cloudy structure; and at thickness counting approximate 80μm, we could harvest high-quality consecutive sections. The result of HE staining turned favourable, the microscopic histological structure were intact, cell nucleus and plasma were fresh; and there was also less section tissue fading during immunohistochemistry staining. Conclusion Perfusion fixation, time of dehydration and clearing, selection of waxdip and embedding, temperature of flattening water bath and the use of adhesion slides, are the key factors to the preparation of high-quality successive paraffin sections of rat brain tissue.