Thermoregulatory Tracing Part III: ISH

In situ Hybridization is the final step of the experiment, but it is also the hardest step.  Although there is a basic outline for ISH protocols, each protocol is customized for the specific probe.  This customization is largely a matter of trial and error, which can be extremely frustrating and time-consuming.  This issue is further compounded by the fact that ISH protocols require three full days to run before any results are seen, making it very possible to make mistakes and thus waste a lot of time and effort.

The ISH protocol essentially rinses the tissue of interest in several permeabilizing solutions that open the cell so that the probe can penetrate.  After the cells have been permeabilized, they are put into a solution containing the probe so that the probes can bind to the RNA.  After the probe insertion, the tissues go through several rinses to ensure that no probe is leftover that could cause a false positive.  The third day is when the antibody to DIG is inserted to cause the color reaction, which ultimately allows the probes to be visualized.

My research is currently in the ISH phase of the experiment.  We have tried several different protocols with no luck, but I am still optimistic that it will eventually work.  I am currently experimenting with different tissue sizes and buffer concentrations to see what will work best in the protocol.  Once I find an ISH protocol that works, it will be smooth sailing from there.  It will be relatively easy to test the efficacy of the gold nanoparticles and to visualize the glutamatergic pathways in the hypothalamus.  I am looking forward to starting my research up once again this semester and finally reaching a conclusion on this project.