PCR analysis of 70-μm-thick tissue samples, microdissected cell clusters, and microdissected single nuclei of α-actin–positive neointimal VSM cells after allogeneic aorta transplantation. (a) Tap2 PCR analysis of 70-μm-thick tissue samples from aortic allografts transplanted in the BN to Lew, Lew to BN, and Lew to BN + CsA combinations. Lew-derived DNA is cut after HindIII restriction, whereas BN-derived DNA remains uncut. Restriction analysis indicates recipient origin in untreated allografts. After CsA treatment, restriction analysis indicated donor origin. (b) HY PCR analysis of microdissected neointimal cell clusters from allografts transplanted in the female PVG to male AO strain combination. Grafts were 50 Gy γ-irradiated prior to transplantation or left untreated. Representative results of first- and second-round PCR products (549 and 128 bp) of two irradiated and three untreated grafts are shown. All samples show the male-specific DNA fragments after first- and second-round PCR. (c) Micrograph (double staining for α-actin and elastin) on an aortic allograft after isolation of single nuclei using a micromanipulator. Arrowheads indicate remaining gaps after isolation of nuclei of three separate α-actin–positive neointimal cells. Note absence of VSM cells in the media. a, adventitia; m, media; ni, neointima. Asterisk shows an α-actin–positive VSM cell. Original magnification: ×400. (d) HY PCR performed on eight single nuclei microdissected from two aortic allografts transplanted from female PVG to male AO recipient rats, showing the male-specific (recipient-specific) 128-bp DNA fragment after nested PCR. bp, 100-bp ladder; H₂O, water control (i.e., no template DNA present in the PCR reaction mixture).