Chapter Characterization of CD38 Expressed in Mitochondia from Murine Brain A * * B * * Figure 4.15 CD38 immunoreactivity in mouse cerebellum, labeled with goat polyclonal CD38 antibody-sc-7049. The immunoreactivity was present in many dendrites. The postsynaptic densities showed the most intense labeling (arrow head) followed by CD38 staining associated with mitochondria. Minimal labeling was observed alongside the plasma membrane (B). Negative immunostaining observed in CD38 KO samples (A). -Axon terminal. white arrow-CD38 immunoreactive postsynaptic membrane. Scale bar: 0.2µm * 188 Chapter Characterization of CD38 Expressed in Mitochondia from Murine Brain A B Figure 4.16 CD38 immunoreactivity in mouse cerebellum, labeled with goat polyclonal CD38 antibody-sc-7049. The labeling occurred diffusely throughout the processes at the granular layer (A), in particular association with mitochondria in varying degree of CD38 staining intensity (B). Notice the the immunostaing is restricted to surface of mitochondria (arrow). Scale bar: 0.5µm 189 Chapter Characterization of CD38 Expressed in Mitochondia from Murine Brain Gd Figure 4.17 CD38 immunoreactivity in mouse cerebellum, labeled with goat polyclonal CD38 antibody-sc-7049. Intense immunolabeling was associated with mitochondria, plasma membrane in the Golgi cell dendrite region (Gd) at molecular layer. Varying degree of CD38 immunostaining associated with mitochondria in different region was observed (arrow). The postsynaptic densities showed the most intense labeling (arrowhead). Scale bar:1µm 190 Chapter Characterization of CD38 Expressed in Mitochondia from Murine Brain 4.2.4.2 Localization of CD38 on mitochondria using immunogold labeling Following the EM/immunoperoxidase study as mentioned above, independent confirmation of CD38 localization to the outer mitochondrial membrane was sought by EM/immunogold labeling experiments. This has a direct advantage over immunofluoresence or immunoperoxidase in that the labeling of individual samples can be correlated with ultrastructural features, such comparisons are important because the EM/immunogold technique has an excellent signal to noise ratio with practically no background noise, making the categorization of cells as + or – staining absolutely unambiguous (de Harven et al., 1993). True enough, immunogold labeling does indeed further confirm the results obtained from immunoperoxidase study. Immunogold localization of CD38 using a 15nm gold conjugate rabbit antigoat IgG revealed the presence of CD38 in isolated mitochondria from the wild type mice brain tissues. The result was examined with the use of sample blanks. In the sample blanks, negative controls, a) Non-immune Goat IgG instead of primary goat CD38 antibody and b) isolated mitochondria from CD38 KO mice brain tissues were employed to examine for the specificity of the CD38 antibody in this experiment (data not shown). As expected, there were no gold particles detected in association with the purified mitochondria isolated from CD38 KO mouse brains. In electron micrographs of isolated mitochondria from the wild type mice brain tissues, the gold labeling clearly showed that CD38 is indeed present on the mitochondria. The staining was consistently observed on the outer aspect of the outer membrane of mitochondria exposed to CD38-specific primary antibody, sc-7049 (Figure 4.18 (A) and (B)). The pattern of CD38 distribution heterogenous, as illustrated in the images presented. The background noise level of the immunogold staining in the electron micrographs was low in all the sections observed. 191 Chapter Characterization of CD38 Expressed in Mitochondia from Murine Brain In comparison with the immunoelectron microscopy studies of CD38 localization on mitochondria by immunoperoxidase labeling with DAB, the results obtained from the immunogold labeling of fractionated mitochondria showed consistent CD38 labeling and distribution pattern on the surface of mitochondria. This also showed that the fractionated procedure did not alter the localization of CD38 on the organelle. These data further supported the localization of CD38 on the outer mitochondrial membrane which strengthens the interpretation that the association of CD38 with the mitochondria is not due to the extraction procedure. Altogether, these results showed specific localization of CD38 on the outer mitochondrial membrane and a specific orientation of the molecule present on the mitochondria. Immunogold localization of nicastrin, an inner mitochondrial resident membrane protein was employed in this study to serve as a comparison to the outer mitochondrial membrane localization of CD38. Here, it was observed that most gold particles labeled the interior of mitochondria; only a few gold particles were on or near the rim of mitochondria (Figure 4.19 A-C). The electron micrographs obtained showed the pattern of internal staining, as opposed to the pattern of staining with CD38 involving the surface of the outer mitochondrial membrane facing the cytosolic side. In contrast to the immunogold labeling of CD38 on the outer mitochondria membrane, all gold particles observed were distributed throughout the interior of mitochondria in close association with the inner mitochondria membrane. None of the gold particles was found associated on the outer mitochondrial membrane facing the cytosolic side. 192 Chapter Characterization of CD38 Expressed in Mitochondia from Murine Brain A B Figure 4.18 Immunoelectron microscopy of CD38 localization in mitochondria isolated from mouse brains, labeled with goat polyclonal CD38 antibody-sc-7049. AB, representative images of mitochondria isolated from mice brain stained with antiCD38 antibodies (gold particles are indicated with arrows). These images represent different patterns of abundance of gold particles associated with the cytoplasmic face of the outer mitochondrial membrane. Scale bar: 0.2µm 193 . micrographs obtained showed the pattern of internal staining, as opposed to the pattern of staining with CD38 involving the surface of the outer mitochondrial membrane facing the cytosolic. particular association with mitochondria in varying degree of CD38 staining intensity (B). Notice the the immunostaing is restricted to surface of mitochondria (arrow). Scale bar: 0 .5 m B A . head) followed by CD38 staining associated with mitochondria. Minimal labeling was observed alongside the plasma membrane (B). Negative immunostaining observed in CD38 KO samples (A) . -Axon