Monday, July 22, 2013

At the heart of the matter: an introduction to transcardial perfusion

WARNING: This post describes a rodent non-survival surgery. Reader discretion advised.

This week, I was able to observe Feng performing transcardial perfusion, a technique used to prepare tissue for immunohistochemistry.

The subject animal (in this case, a mouse) is deeply anesthetized before surgery is performed to expose the heart.

Gage, G. J., Kipke, D. R., Shain, W. Whole Animal Perfusion Fixation for Rodents. J Vis. Exp. (65), e3564, doi:10.3791/3564 (2012).

A perfusion needle is placed in the left ventricle (the chamber of the heart responsible for pumping blood to the rest of the body), and a saline solution is then pumped at constant pressure through the circulatory system before exiting through an incision made to the right atrium. This clears the subject of blood.

Gage, G. J., Kipke, D. R., Shain, W. Whole Animal Perfusion Fixation for Rodents. J Vis. Exp. (65), e3564, doi:10.3791/3564 (2012).

A paraformaldehyde (PFA) solution is then accordingly pumped through the cleared circulatory system, quickly and consistently preserving the tissue in a life-like state.

Gage, G. J., Kipke, D. R., Shain, W. Whole Animal Perfusion Fixation for Rodents. J Vis. Exp. (65), e3564, doi:10.3791/3564 (2012).

The fixed tissue can then be harvested and stored for later experiment. For our experimental needs, whole animal perfusion fixation yields the best possible preservation of the brain for immunohistochemistry.

Gage, G. J., Kipke, D. R., Shain, W. Whole Animal Perfusion Fixation for Rodents. J Vis. Exp. (65), e3564, doi:10.3791/3564 (2012).

Through my observation, I learned that safety and sterility, for both the animal subject and the human investigator, are of the utmost important in any procedure of this nature. Due to the toxic characteristice of PFA, all work is done under the fume hood, and gloves and a lab coat are donned at all times. And because our lab works with both live tissue and fixed tissue, we have a special set of tools put aside for each, to ensure that no cross-contamination occurs.

It was a very good learning experience, and I enjoyed helping with the set-up and clean-up for the procedure. I'm sure that as Laura begins to undertake her own immunohistochemistry experiments in the future, I'll be assisting with many more perfusions.

Tuesday, July 16, 2013

Spirit check

In today's post, I'm taking a break from the lab and sharing with you a different facet of my life . . . dancing!

I took my first ballet class when I was three years old, and since then, I've fallen in love with dance both as a sport and as an art form. Dance practice is the space in my busy schedule where I can clear my mind of everything else going on in my life and just focus on my body's movement.

In the spring, I auditioned for UH's Cougar Dolls Dance Team and made it onto their 2013-2014 squad!

The last night of auditions when we received our roses! I'm second from the right in the back row.

This weekend, we travelled to Texas State University in San Marcos to attend UDA (Universal Dance Association) College Camp. We performed as a team for the first time, learned some awesome new choreography to bring back to UH, and got to interact with other college dance teams from all over the country.

Representing the Houston Cougars at fight song evaluations! I'm second from the right in the bottom row.

Overall, it was an amazing experience. We really bonded as a team, and we're now looking forward to football season when we'll get to cheer on our boys at Reliant Stadium!

Keep up with the Dolls at our Facebook page. Go Coogs!

Monday, July 8, 2013

Rabbits and goats and guinea pigs . . . oh my!!

This past week was rather short due to the 4th of July holiday, and my behavioral tests are now postponed indefinitely. :(

But no matter! I am just as happy helping Laura design her future experiment. Last Tuesday, she gave me a crash course in immunohistochemistry (IHC). I also found a neat video that really clarified the technique for me. Watch it here.

We'll be using indirect IHC to visualize a number of inflammation markers in rodent hippocampal brain slices. But instead of using a marker like DAB (as seen in the video), we'll use fluorescent tags on our secondary antibodies that can be imaged with the confocal microscope.

Right now, we're looking at five different primary antibodies - one for each target molecule - from five different host animals: goat, guinea pig, rat, mouse, and rabbit. So many animals!

I'm also having fun playing with Life Technologies' Fluorescence SpectraViewer to map out which fluorophores we want to use with our secondaries. It is important that each tag's excitation and emission wavelengths are unique to prevent crosstalk and to produce a clear image.

Good example, adequate spacing.
Bad example, overlapping signals.

Try it out yourself!