Don’t Throw Stones in a Glass-Bottomed Dish

We all know the old saying, “Don’t Throw Stones in a Glass House”…. Well, those same principles apply to axotomies in glass-bottomed Petri dishes. After tedious preparation of a zebra finch DRG culture in a 35mm plate, I was ready to image calcium fluctuations and Wallerian degeneration on the confocal microscope. All of the necessary ingredients had been added: Fluo-4 to stain the calcium under FITC illumination, EDTA to chelate the extracellular calcium, and FDUR/NGF to continue growth. This was going to be our first experiment in the live cell chamber with controlled CO2, temperature, humidity, and air flow. The glass bottom of the dish was allowing for clear resolution on the pre-axotomy pictures and I was ready to induce injury on the axons.

I had marked five locations where I was going to cut and track calcium spikes with a time-lapse acquisition. The first two cuts went well but unlike plastic wells, the glass did not show a clear marking where the axotomizer had made contact. Thus, to ensure I had cut the axon entirely, I pushed a little bit harder. All of a sudden, the glass circle in the bottom of the dish came unglued and detached from the rest of the well. Needless to say, the experiment was ruined.

This was the first truly unfortunate failure of the summer for my research but to ameliorate the situation, I will repeat the experiment on a plastic Petri dish and sacrifice a little bit of quality for the sake of never recreating that situation again. Thank you for listening to me complain for an entire blog post about my glass-bottomed debacle. Next blog post, I will talk about collecting data from the calcium spikes with ROI’s and histograms!