Friday, June 15, 2012

Week 4-Dealing with Collected Tissue

After tons of paper-writing, peer editing, and data-analyzing from the previous week, this week required much more activity. Some of the mice from our experimental groups finally reached 21 weeks of age and were ready to get their blood pressure & heart values confirmed by an invasive procedure dealing with the carotid artery. With this procedure completed, it was time to carefully stain the collected aortas in order to conduct lipid lesion formation analysis.

First, I drained the aortas with a 100% propylene glycol solution. This dehydrates the tissue and prepares them for staining. Next, I incubated the aortas with the stain for 4 hours at room temperature. After the 4 hours were completed, I washed the aortas with 85% propylene glycol solution. Last, I incubated the aortas overnight with a PBS (saline) solution in order to preserve the tissue and prevent it from drying out.
Collected Tissue 
The Staining Process 
100% Propylene Glycol

Collected Tissue

PBS Solution
In addition to this process, I learned how to filter the substrates to make the stain solution! This required a biosafety cabinet and a fume hood to catch the vapors of this potentially hazardous substance. This process also requires the use of a vacuum to help filter the substrate from the liquid. With a special device that connects to the top of the bottle, the vacuum acts on the substrate found in the upper portion of the device and drains all of the liquid to the bottom jar where it is then collected and stored. Because the stain is light-sensitive, we made sure to cover the bottle in aluminum foil to prevent the light from changing the chemical properties of the stain.

Filtering Oil Red O

Oil Red O Solution-Final Product

Other activities this week included washing the membranes in ongoing Western Blots, switching out the PBS solution that was used to store the collected aortas, prepration of TBST & milk, and storage of membranes from Western Blots into sealed packs.
Washing the Membranes 
Incubating Western Blot

Membrane from Western Blot


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