Immunolabeling controls

Incubate in affinity-purified rabbit anti-cannabinoid receptor IgG overnight at 4°C (in a humidified chamber). A first immunolabeling should include a titration series of the specific affinity-purified antibody at dilutions of 1 5-1 20 to establish the optimal dilution. This titration series is required to optimize the signal-to-noise ratio of immunolabeling. Concnrrent incubations should be carried out in non-immune rabbit IgG (at similar dilutions) from the primary antibody donor species to serve as negative controls. 

Fig. 5. Immunogold labeling for the demonstration of the human CBj cannabinoid receptor as expressed on the envelope of a baculovirus CB2 expression vector in insect cells. (A) Intense immunogold label surrounds the envelope (arrow) of the baculovirus construct. (B) Control P-galactosidase recombinant baculovirus envelopes show no immunolabeling for the CB2 cannabinoid receptor. Scale bars, A and B, 0.2 jm.

Fig. 8 Confocal fluorescence microscopy of B50 neuroblastoma cells, (a, b) Immunolabeling of mtHSP70 green) in B50 cells in controls (a) and cisPt-treated cells (b). After cisPt, mitochondria are clustered around the nucleus and form dense masses in the cytoplasm (b). Nuclei are counterstained with Hoechst blue), (c, d) Double immunolabeling of filamentous actin red) and a-tubulin green) in B50 control cells (c) and in 48 h cisPt-treated cells (d). CisPt-induced cytoskeleton damage leads tubulin to reorganize into thick bundles (e) and to disruption of filamentous actin microfilaments and accumulation of depolymerized actin at cell periphery (f).

Fig. 8 (continued) (g, h) Immunolabeling of Golgi apparatus red) in control cells (g) and in 48-h cisR-treated cells (h). In control cells, the Golgi apparatus maintains its perinuclear location, whereas in late apoptosis cell, the Golgi is redistributed into the cytoplasm. DNA is counterstained with Hoechst 33258 (blue). Scale bars 20 pm... 

AIF is an apoptotic effector protein that induces chromatin condensation and DNA fragmentation [51], Confocal microscopy (Fig. 9) reveals that AIF is strictly confined to mitochondria and thus colocalizes with mtHSP70 in control cells (Fig. 9a). In early apoptosis, AIF translocates to the nucleus, but in late apoptosis (with fragmented chromatin), immunolabeling moves back in the cytoplasm again in mitochondria [52-54], After treatment with cisPt (Fig. 9), AIF and mtHSP70 colocalize in clusters within the perinuclear cytoplasm. 

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