Chapter Characterization of CD38 Expressed in Mitochondia from Murine Brain A B Figure 4.19 Immunoelectron microscopy analysis of Nicastrin localization in mitochondria from mice brain tissue. A-C, representative images of mitochondria from mice brain stained with anti-Nicastrin antibodies. arrow- outer mitochondria membrane on the cytosolic site. Scale bar: 0.2µmm C 194 Chapter Characterization of CD38 Expressed in Mitochondia from Murine Brain 4.2.5 Determination of the localization of CD38 on mitochondria using Scanning Electron Microscopy (SEM) 4.2.5.1 Determination of the purity of mitochondria by scanning electron microscopy (SEM) Further confirmation of the purity of the Percoll purified fractions can be observed in Figure 4.20. Figures 4.20 (A) and (B) show ISEM (immunoscanning electron micrograph) of crude mitochondria and Percoll purified mitochondrial fraction. By comparing both Figures 4.20 (A) and (B) it is evident that both mitochondrial fractions contain intact mitochondria with an outer membrane surrounding the distinctive cristae comprising electron-dense packed inner membrane-matrix compartment. The crude preparation of the mitochondrial sample solely by differential centrifugation or prior to Percoll purification showed extensive contamination by non-mitochondrial components including circular membranous structure mainly derived from cell membranes, microsomes, myelin, small vesicles and other cellular debris (Figure 4.20A). The crude mitochondria also contained ghost-like membranes of various sizes associated with mitochondrial fraction in Figure 4.20 (A). A similar observation was not made with the Percoll purified mitochondrial fraction in Figure 4.20; (B) instead it showed highly purified mitochondria devoid of microsomal contamination. Seen on electron micrographs obtained from SEM, purified mitochondria were found to be in close association with each other and the mitochondrial cristae were clearly distinguishable in the matrix display tubular cristae with lumen in continuity with space between the inner and outer mitochondrial membrane. The predominant morphology was varying size of mitochondria with dense cristae with varying shapes of round, tubular to spherical (Figure 4.20B). 195 Chapter Characterization of CD38 Expressed in Mitochondia from Murine Brain Mitochondrial samples selected for analysis by ISEM were coated with a layer of carbon rather than an alloy such as gold/palladium. Consequently, the resulting images, generated by backscatter and secondary electrons, lacked the quality normally characteristic of samples prepared for ISEM. The phenomenon of charging was evident along the surface of many samples. Despite this difficulty, identification of 15nm colloidal gold particles was still possible (Figure 4.21-4.23). The consistent presence of identifiable colloidal gold particles in observed mitochondria samples indicates the potential of this approach to analyze the distribution of antigenic determinates on a three-dimensional level. Specifically, ISEM would allow the examination of the localization and quantification of molecule on the surface of mitochondria. 196 Chapter Characterization of CD38 Expressed in Mitochondia from Murine Brain A B Figure 4.20 Backscatter electron micrographs of mitochondrial fractions, labeled with goat polyclonal CD38 antibody, sc-7049. The probe were rabbit anti-goat conjugated with 15nm colloidal gold particles. (A) Crude mitochondrial fraction (B) Percoll purified mitochondrial fraction. Scale bar: 1µm 197 Chapter Characterization of CD38 Expressed in Mitochondia from Murine Brain A B Figure 4.21 Backscatter electron imaging (BEI) of mitochondrial fractions, labeled with goat polyclonal CD38 antibody-sc-7049, and viewed at high magnification. The probe were rabbit anti-goat conjugated with 15nm colloidal gold particles as described in Materials & Methods. The image is showed at low magnification from the experiment with the labeling of CD38. The insets (A and B) are enlargement of the encircled areas of the isolated mitochondria. (A) shows sample viewed in the BEI mode (reverse polarity). All the gold particles appeared in good contrast over the surface of the mitochondria, with the surface structures showing distinguishable clear cristae structure. (B) same sample viewed in the same field as in (A), viewed after mixing the SEI and the BEI (normal polarity) signals. The gold particles were still vividly seen and superimposed to the image of the mitochondrial surface. Scale bar: 1µm, 100nm 198 . microscopy analysis of Nicastrin localiza tion in mitochondria from mice brain tissue. A- C, representative images of mitochondria from mice brain stained with anti- Nicastrin antibodies. arrow- . Chapter 4 Characterization of CD38 Expressed in Mitochondia from Murine Brain 1 96 Mitochondrial samples selected for analysis by ISEM were coated with a layer of carbon rather than an alloy. purified mitochondrial fraction. By comparing both Figures 4.20 (A) and (B) it is evident that both mitochondrial fractions contain intact mitochondria with an outer membrane surrounding the distinctive