Fig. 1

Visualization of Casp3 activation in fixed OCCs after biolistic transfection. a Low magnification image of a double-transfected OCC (pSCAT3-DEVD + pHcRed1-C1) after excitation with the 588 nm argon laser line. HcRed1 expression permits an easy visualization, localization, and identification of successfully transfected cells. The red-dotted line indicates the border of the culture. b-g Exemplificative images of two CGCs in the IGL (b-d) and two CGCs (e-g) in the EGL after pSCAT3-DEVD transfection showing the emissions of the FRET pair at 475 nm (ECFP) and 530 nm (Venus). The cell at right in b-d is a CGC in the vertical bipolar stage of migration and displays a well visible axon (asterisk) that bifurcates to give origin to a parallel fiber. The two cells in e-g are CGCs at the horizontal bipolar stage of migration. The cell at right displays some enlargements of its processes with high Casp3 activity (arrowheads). Note that to better show the distribution of ECFP and Venus images are taken at different laser excitation powers. As an example, the true fluorochrome emissions during FRET recording are shown in black and white in the inserts of panels b and c. In d and g cells are imaged in pseudocolor using a logarithmic scale to display the ECFP_em/Venus_em ratio. Note the cellular resolution of the FRET probe. h Combined ICC for the marker NeuN (green channel) and biolistic transfection with pHcRed1-Surv (red channel) shows two transfected CGCs in the IGL. Both cells are in the vertical bipolar stage and their axons have been labeled by the asterisks. Image has been modified and reproduced with permission from [29]. i-k Combined ICC for cCasp3 (red channel) and biolistic transfection with pSCAT3-DEVD (green channel) after induction of apoptosis with 1 mM NMDA for 48 h shows several cCasp3 immunoreactive cells. The pattern of cellular localization of the 17/19 kDa fragment of the protease is different among cells, one of which (arrow) displays a highly condensed cCasp3 positive nucleus. The larger cell transfected with pSCAT3-DEVD displays cytoplasmic cCasp3 immunoreactivity, but the nucleus (arrowhead) is negative. Abbreviations: cCasp3 = cleaved caspase 3; CGC = cerebellar granule cell; ECFP = enhanced cyan fluorescent protein; EGL = external granular layer of forming cerebellar cortex; IGL = internal granular layer of forming cerebellar cortex; NeuN = nuclear nuclei antigen; NMDA = N-methyl-D-aspartate; Surv = survivin; Venus = mutated yellow fluorescent protein. Scale bars: A = 500 μm; b-k: 10 μm