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The guide cannula measured 2.5 cm in overall length and was prepared with a heat bulge placed 2.5 mm from its beveled tip, thus acting as a stop to control the depth of penetration

At this time animals were not kept in germ-free environments

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At 0, 3, 7, and 10 days, the tissues were fixed, and cryosections were used for in situ apoptotic staining with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), according to the manufacturers instructions (Servicebio, Beijing, China)