Before the data collection...

If you are going to ask me to sum up my research project in one sentense, it would probably be something like this: using fluorescence antibody to study whether the location/trafficking of the Na+/K+-ATPases differ between a control locust and a locust that has received a heat-shock pre-treatment in their meta-thoracic ganglia (a pile of neurons enclosed by a layer of sheath). Sounds pretty easy you say? Yeah, that's what I thought too, before I started my experiments...

I've learned couple of things in the past year I've spend on doing my Master’s. For example, your experiment always take much longer than what your expected or as indicated on your protocol. But an even more important lesson I've learned was if this is the first time your lab doing a type of experiment, the chances are, it probably will not work at the first try;

My experiment sounded simple enough, dissect out the ganglia, incubate them with antibodies, and then study them through a confocal microscope; but the reality was far from that...

Trial 1:

Like a kid going to his first day of school, I was very excited about starting on my immunohistochemical experiments. I did my research and came up with a feasible protocol. I followed the protocol word to word, incubating the ganglion in antibodies  (red fluorescence) to stain the ATPases and DAPI  (blue fluorescence) to stain the cellular nuclei. Once the samples were ready, I just could not wait to see what they would look like under the confocal.

But to my least expected, this is what came up under the confocal... At first, I had no idea what was going on. But after my supervisor has looked at the images, we concluded only DAPI was small enough to penetrate the dense shealth that is enclosing the ganglia. Consequently, the sheath blocked out all the antibodies, resulting in only those visible cellular nuclei stained by DAPI. The red that we are seeing in our images were the visible antibodies that were the residues trapped inside of the ganglia trachia.

Well, I guess my first trial didn't work, looks like it's time for me to do more research and back to the drawing board again...