Hannah in Japan II: Immunohistochemistry and my first trip to Kyoto
Kon'nichiwa!
For the past two weeks at EHUB, I have been working on histology and microscopy. I performed immunohistochemistry staining on tissue that I will use for data collection. The staining process takes several days, as the tissues need to incubate in both primary and secondary antibodies.
On the first day, we start by washing the tissue in a phosphate-buffered saline (PBS) solution to rinse off chemicals used for tissue preservation and prevention of bacterial growth. We then use hydrogen peroxide and skim milk to block any non-specific proteins from being targeted in the staining process. Finally, the tissues are incubated in a primary antibody overnight. Antibodies are Y-shaped proteins that bind to specific antigens (proteins located on the cell). The tissues are later incubated in a secondary antibody, which will bind to the primary antibodies, to amplify the signal during the staining process.
For the targeted cells to be visualized with a color change, we attach a specific enzyme to the secondary proteins using an ABC kit (Avidin-biotin complex). We then submerge the tissue in a chromogen, such as DAB (Diaminobenzidine). Finally, we add hydrogen peroxide to the DAB solution. This chemical interaction causes the DAB to oxidate, resulting in a color change. The tissue will gradually darken in areas where the secondary antibody had attached to the antigen of the target neurons.
In my study, the antigens of interest are GFP (green fluorescent protein) and RFP (red fluorescent protein). These antigens were introduced via a retrograde tracer, which is a virus that is taken up by the axon of a neuron and brought back to the neuron body. The virus is then continually passed between neurons of a specific pathway, until they make their way back to the beginning of this network. In this case, the virus was introduced into the striatum, and the neurons in this area relayed the virus to the cortical and subcortical neurons involved in the corticostriatal network. We then employed anti-GFP and anti-RFP antibodies to locate the neurons labeled with GFP and RFP, respectively.
Before we could stain the tissues, they were first sliced with a microtome into 50-micrometer sections (roughly the width of a human hair) so that they can be properly mounted on a slide and observed through a microscope. During this process, dry ice is used to freeze and harden the tissue, since the blade could tear it easily if it gets too soft. We then use a microscope program called Neurolucida (MBF Bioscience) to plot the distribution of cortical and subcortical neurons in the sections.
Outside the lab
Dakota Smallridge (another KSU student at EHUB) and I were invited with a few others to have dinner with Dr. Tanaka, a professor of primate genetics. He made us takenoko-gohan (bamboo shoot fried rice) with bamboo shoots that he had collected around campus! Dr. Tanaka also showed us how to make onigiri (rice balls) with the leftovers. After dinner he took up his ukulele and played a few songs from the Studio Ghibli movie My Neighbor Totoro!
Over the weekend, I took a shinkansen (bullet train) to Kyoto to meet with Samantha Magrini and Scott McKinny, two of the other students in my program who are conducting research at the main campus. We had lunch at a ramen restaurant and then walked to Heian Shrine, where we toured the gardens and took many photos. The next day we visited Nishiki Market, a street lined with over a hundred shops, restaurants, and food stalls, where we enjoyed many new and delicious foods. We also visited the Gion district of Kyoto, an area historically famous for geisha. Before I left Kyoto, Sam, Scott, and I ate dinner at a shabu-shabu restaurant, where you cook thinly sliced meat and vegetables in a hotpot at your table. It tasted amazing!
Ja ne! (See ya!)
Hannah
This opportunity to conduct research in Japan is supported by NSF-IRES Award #1853937 to Drs. Tosi, Raghanti, Meindl, and Lovejoy at Kent State University.