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Jane Richardson; One Down, One To Go...

We finally did it: an optimized IHC protocol. Peering into the microscope to see the bright red below was an amazing feeling. The SERT was glowing like a Christmas tree. Our procedure incorporated two new elements that helped us achieve the finished procedure: pre-heating of the slide and antigen retrieval. Before beginning the slide washes, we heat the slide in a 55o C water bath. This makes the slide more susceptible to the treatments we use. To complete antigen retrieval, we place the slides in .1 M citrate buffer and then heat it, using a 100o C water bath. This exposes epitopes that were covered in the making of the slide. Therefore, the tissue is more susceptible to antigens. With these two new steps, we were finally able to see the SERT, in Cy3, and the background staining, in DAPI. However, one pitfall of our microscope is we can only look at one contrast at a time. This forces us to take separate pictures of each contrast and overlay them in photo editing (picture of infected ileum attached). Though this works, it is not as effective to accurately see the SERT in the tissue. I am still continuing to complete our procedure, but we need to see the slides on a different microscope. We are planning to conduct training with a more complex microscope that would give us a clearer image. This new microscope is also more accurate because it is enclosed so other factors cannot affect the slides, such as air conditioning and moving particles.
Additionally, this Friday, we put our cDNA samples in the qPCR/rt-PCR machine. Real-time polymerase chain reaction (rt-PCR) allows us to analyze how much of the SERT protein was in each tissue sample at the time of cell death. When another copy of the gene is made, the fluorescence in the PCR tube grows a little stronger. When they reach a certain brightness, copying is stopped. This allows you to compare the amount of SERT in each sample. The faster the sample has reached full fluorescence, the more SERT there is in the sample. Our results were very poor. Typically, you are supposed to see a curve in the graph displayed but our results were jagged and staggering. This meant we had yet another protocol to optimize. Though there is more work to be done finalizing this project, it is not my main focus in the lab, so it will likely take longer to complete. Next time we conduct cDNA synthesis, we plan to use a gel to analyze the genetic material and make sure it is not damaged before we make the cDNA.

This week, I brought cupcakes into the lab from Georgetown Cupcakes. Everyone loved them! The people in my lab are so incredible and extremely helpful. They are always willing to answer my questions when I have them and help me with whatever else I need. I have really enjoyed my time in the lab so far and I am looking forward to the other half of my time here!




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  1. speretzJuly 9, 2018 at 11:59 AM

    Is that your picture??? Beautiful! You should be proud!

    ReplyDelete

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