At this stage of my project, I am staining brain sections with Fluorescence In Situ Hybridization (FISH) and then imaging them. The FISH procedure to stain the mRNA of interest requires one overnight incubation in order for the probes to bind to the mRNA. First, I block and wash the sections. I use salt solutions and buffers to poke holes in the membranes of the cells allowing the probes to enter. I pipette the probes onto the sections with a hybridization buffer and then allow the slides to incubate. The next day I wash the samples and either fix them or proceed with antibody staining.
The next step is imaging the cells. I have signed up to use the microscope for a few hours each day in order to take pictures of the astrocytes. So far I have had difficulties with visualizing the mRNA. One problem is that the neuron specific antibodies block clear visualization of the mRNA. If anyone has any thoughts on how to troubleshoot this problem, let me know. To mitigate the second problem which is unspecific binding of the probes, I performed FISH without using the probes so we could have a control slide. I am working on using this control to be sure that the fluorescent spots I am seeing are really mRNA.
When I am not working on my project, I work on various things for my mentor. Shoutout to Bel my amazing mentor for the summer. She has taught me procedures unrelated to my project from bacterial miniprep to co-culturing astrocytes and neurons. She makes sure I understand how the buffers and reagents work, as well as the overall biological systems the lab is studying. She has also worked with me on every step of my project, from perfusing the mice to harvesting and sectioning their brains to what I am working on now.