Trial 2

After I collected my thoughts back together and got over about my experiment did not work, I concluded that the reasons it failed could either be because 1) the antibodies would not penetrate through the sheath, or 2) the antibodies are not binding onto the ATPase (which I doubted and really hoped was not the case, because from the background research that was the only Na+/K+-ATPase antibody other researchers have used on insect Na+/K+-ATPase. Therefore, I didn't have much choice in terms of choosing antibodies). After discussing the problem and possible solutions with my supervisor, he suggested that I could use paraffin and sectioning to mount the tissues in wax and section the ganglia into slices to eliminate the problem created by the sheath. Therefore, if this experiment worked, then we can conclude that the first time my experiment didn't work was because the antibodies weren't able to penetrate the sheath and rule out the possibility that the antibodies are not binding on to the ATPase.

After I have my ganglia sectioned by a lab technician from another department, paraffined and stained, I'm ready for round 2 of confocal. This time the staining worked (the green fluorescence),  indicating that antibodies were able to enter the cell body and stained the ATPase. But as one problem is solved, another problem emerged. Because of the fragility and the size of a ganglion, which is roughly the size of a pen's tip, the ganglia were heavily damaged during the sectioning process and orientated in a random fashion. As a result, we were not able to identify the neurons within each ganglion and to make valid comparison between control and heat-shocked ganglia. Consequently, it appears that paraffin and sectioning also may not be the route to go about my experiment. Now, I will need to come up with some other method that does not involve sectioning but yet still allows the antibodies to penetrate through the sheath...