Acetylcholinesterase Tissue Staining

The last staining step was targeting acetylcholinesterase, which is an enzyme that catalyzes the hydrolysis reaction of acetylcholine. We did this similarly to the parvalbumin staining, with a few tweaks to the protocol. First, the solution was prepared with a phosphate buffer and hydrogen peroxide. Then, a maleate buffer was used, which caused a slight peach color during incubation. TRIS Buffer rinses were followed by an incubation period , which was then followed by hydrogen peroxide and Tris Buffer rinses. The tissues from the three rat brains successfully reached a dark purple to black color, and were then mounted on gelatinous slides. These slides as well as the parvalbumin stained tissue slides will be subsequently analyzed by Dr. Burk to assess the effectiveness of  anti-ChAT saporin as a novel immunotoxin in the thalamic reticular nucleus. 

Algae Progress

Another aspect of my work this summer is obtaining calorimetric values for samples of mainly algae and hydroids.  I am using a Parr 6400 calorimeter, which shows the amount of heat energy contained within a sample in terms of calories per gram.  The top performing algal samples thus far are a type of red algae with an average output of 2052.09 cal/g, while the hydroid samples are our overall top performer with an average output of 3075.88 cal/g.  The algal samples have trouble igniting by themselves in the calorimeter and so a spiking substance, solid benzoic acid, is added as an accelerant.  The calorimeter that I am using is able to account for the heat produced by the benzoic acid and subtract that amount so that the “gross heat” figure given by the calorimeter concluding a test is the heat energy released by the sample.  I would also like to test samples of Enteromorpha before the end of this session.  The other side of this project has been growing algae on our craft and designing/implementing a pulley system to raise the screens on which the algae is growing out of the water for collection and maintenance.  At the moment this craft is still in the boat basin at VIMS but will be deployed before this session is over.

The Home Stretch: Finalizing Data and Analysis

Hello again. As the summer approaches its conclusion, I seek to approach a data conclusion of my own. This summer so far has been full of successes and failures alike. From the fiasco when my glass-bottomed dish that had promising results on it broke mid experiment, to improving our cultures to the point where we can consistently grow long, singular axons across most of a well, the spectrum of success has been quite distributive. As I type, I am preparing my grand finale experiment of using a 35 mm dish with five DRG explants on it. The axons have had time to grow for two days and look ready for an axotomy, calcium staining, and imaging. I will, once again, use the live cell chamber on the confocal microscope to simulate physiological conditions in the body. Hopefully, this will more accurately and credibly yield data that mimics what would happen in the body of a zebra finch during axon degeneration.

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Data Analysis Cont.

It is unbelievable that the research period is just about over already! The past 8 weeks have flown by. However, progress with data analysis is slowing down. I have successfully cleaned all my insect data and calculated several diversity measures for each site and transect using R for Statistical Analysis. Using R was not as terribly daunting as I was anticipating though it was frustrating at times. The feeling of triumph when a code would finally work made the frustration well worth it! Altogether, I have calculated abundance, species richness, Simpson’s diversity index, Chao’s species estimator, Pielou’s evenness measure, and rarefaction curves for each data site. I am now working on turning my calculations into visuals. I can preliminarily judge the differing diversities of the sites, but will also be running statistical tests on the data to see which sites are overall “more diverse”.  I will obtain the patch sizes for each transect to complete my analysis.

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The Flowing Afterglow

Aside from my main project, I have been helping get another mass spectrometer in the lab up and running: the flowing afterglow.  This instrument allows us to conduct reactions in the flow tube to produce ions using volatile compounds.  In addition, temperature and pressure can be controlled in the instrument to create an atmospheric environment for these reactions.

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