zeiss colocalization procedure  (Carl Zeiss)

Journal:

Article Title: Met-HGF/SF Signal Transduction Induces Mimp, a Novel Mitochondrial Carrier Homologue, Which Leads to Mitochondrial Depolarization 1

doi:

Figure Lengend Snippet: Mimp is localized to the mitochondrial membrane. (A) CLSM analysis of NIH-3T3 stably transfected with HA-Mimp stained with anti-HA antibody (a) and with MitoTracker Red (b). For colocalization analysis, the green and red images were overlaid (c). The yellow staining represents regions of colocalization. As a negative control, cells transfected with a control vector were stained with anti-HA antibody (a0). (B) CLSM analysis of NIH-3T3 cells stably transfected with Mimp-GFP (a) and stained with MitoTracker Red (b). The colocalization image is shown in (c). (C) CLSM analysis of 293T cells transiently transfected with Mimp-GFP (a) and stained with MitoTracker Red (b). For colocalization analysis, the green and the red images were overlaid (c). Graphic depiction of pixel intensities of image was performed (d). A region of maximal colocalization was selected in the graph (circled in red) and the pixels with colocalized values are represented in blue (e) and overlaid on the colocalization image (f). (D) Western blot analysis of subcellular fractions of Mimp-GFP transfected 293T. HM, fraction enriched for mitochondria; LM, light membrane fraction; S, cytosolic fraction. Mimp-GFP protein was detected predominantly in the HM fraction using an anti-GFP antibody. The same blot was stripped and the mitochondrial fraction detected with an antibody against the mitochondrial-specific protein cytochrome oxidase.

Article Snippet: We performed colocalization analysis using the Zeiss colocalization procedure as described in Materials and Methods section to confirm our observations.

Techniques: Membrane, Stable Transfection, Transfection, Staining, Negative Control, Control, Plasmid Preparation, Western Blot