Microglia in mouse brain tissue: Optimization of immunohistochemical staining using brightfield, epifluorescent, and dual epiflu




Professor Of Neuroscience/anesthesiology and Critical Care Medicine

Project Summary

This summer, I had the opportunity to work in the Eisch Lab at the Children’s Hopsital of Philadelphia under the mentorship of Dr. Amelia Eisch, Dr. Danielle Barber, and Lyles Clark. The Eisch Lab studies neurogenesis in a key brain region, the hippocampal dentate gyrus, which is involved in mood regulation, memory, reward processing, and pattern separation. Specifically, my project was focused on investigating how mild traumatic brain injury (mTBI) impacts microglia in the adolescent mouse dentate gyrus.

Microglia are the primary cellular component of the innate immune system in the central nervous system. Microglia typically survey the brain, playing a crucial role in its response to injury. They become “activated” to help clear cellular debris and contribute to tissue repair following neuronal death. Previous research has demonstrated increased activation of microglia following moderate and severe TBI. However, the microglial response to mTBI has not been extensively studied yet. Additional studies have reported that following injury, microglia can express the protein doublecortin (DCX), a somewhat surprising finding, since this protein is normally only expressed in immature neurons. Using an established model for mouse mTBI and immunohistochemical staining (IHC) for the microglial marker Iba1, the Eisch Lab is interested in studying the microglial response to mTBI, primarily focusing on changes in microglial number and morphology. However, in order to assess these changes, optimal staining parameters for Iba1 need to be initially determined in non-injured tissue.

My project had two main goals. The first was to determine the optimal primary antibody concentration of rabbit anti-Iba1 and goat anti-DCX to be used in single label IHCs for the proteins Iba1 and DCX. The second was to determine whether DCX is a specific marker for immature neurons or also expressed in microglia in non-injured tissue, using double label IHC for Iba1 and DCX.

Being a part of PURM was such an enlightening experience for me. It was so amazing to take knowledge gained within the classroom and see how it is applied in the real-world research setting. Not only did I have the opportunity to learn fundamental laboratory skills including immunohistochemistry and fluorescent microscopy, but through attending Eisch Lab meetings and Neuroscience department presentations, I was also able to see the dynamics of research labs. Additionally, I gained an understanding of the thought processes behind the research we were doing. One really valuable thing I learned is that sometimes, different parts of the scientific process need to be repeated multiple times before they are perfected and can be used in a project. Another crucial thing I learned is how important teamwork is to science, and I feel so fortunate to have spent my summer in such a collaborative environment. I am planning to continue learning and conducting research at the Eisch Lab throughout the school year, and I am so thankful for the PURM program and the Eisch Lab for providing me with such a valuable introduction to neuroscience research at Penn.