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.

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Thermoregulatory Tracing Part II: Probe Synthesis

Once there is a purified sample of DNA, it is time for the next step, probe synthesis.  As I stated before, ISH uses a complimentary RNA probe that binds to the RNA of interest and allows the experimenter to see which cells express that RNA.  In order to visualize the probe, a color reaction must take place.  In this case, the probe contains nucleic acids that are bound to a molecule known as digoxidenin (DIG).  This molecule is incorporated into the probe.  Once the probe has bound to the RNA of interest, the scientist performs a color reaction that involves a colored antibody that binds specifically to DIG, thus lighting up the probes once they are inside the cells.

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Thermoregulatory Tracing Part I: DNA Synthesis and Purification

In situ hybridization (ISH) is a laboratory protocol that takes advantage of nucleic acid base pairing to identify the messenger RNAs present in cells.  Essentially the experimenter makes an RNA probe that is complimentary to the RNA of interest that will produce a color reaction in the cells where the RNA of interest is present.  This protocol is extremely useful because based on the central dogma (DNA –> RNA –> protein), scientists can use it to examine gene and protein regulation in cells.

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Thermoregulatory Tracing: Overview

As I stated in my previous blog, my project involves using a retroactive in situ hybridization (ISH) for the rat vesicular glutamate transporter (VGLUT2) to test the efficacy of the gold nanoprobes that are used in the lab for axonal tracing studies.  Ultimately this project is a stepping stone for future ISH studies in neurons, which will hopefully help to elucidate the thermoregulatory pathways we have been studying.

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Characterization and Tracing of Thermoregulatory Neurons: Introduction

Since spring semester of my freshman year, I have been working in Dr. John Griffin’s lab.  Dr.  Griffin is a neuroscience professor at the college, and his research focuses on determining the thermoregulatory pathways in the hypothalamus.  In general, the lab studies the effects that certain drugs have on the firing rate of neurons to determine what receptors and molecules might be at play in thermoregulation.  Lately, however, I have been working on two new projects that could provide major breakthroughs in the study of thermoregulation.

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