Figure 4.

Immunofluorescence-confocal microscopy and immuno-electron microscopy of FRAP in 3T3 cells. (A) Confocal microscopy shows colocolization of FRAP and mitochondria. MTR was used to stain the mitochondria before fixation and permeabilization. COX-1 was primarily stained with an anti-COX-1 mAb, FRAP was primarily stained with an anti-FRAP mAb, and c-myc was primarily stained with an anti-c-Myc mAb. An [Alexa-fluor-488]-conjugated antibody was used as the secondary stain. The yellow speckles in the overlay frames indicate colocolization. COX-1 shows complete colocolization (positive control for methodology) whereas c-Myc does not colocalize with mitochondria (negative control for methodology). A significant portion of FRAP is seen to colocalize with the mitochondria. (B and C) Immuno-electron microscopy indicates mitochondrial locolization of FRAP. A secondary stain of streptavidin-gold was used to detect the primary anti-FRAP mAb. Arrows point toward the electron-dense secondary probe within the boxes. In C the long arrows point toward typical membrane-proximal localizations that are seen at a high frequency. (Magnifications: ×15,000.)