Trumpets? I don’t even hear bagpipes. Can I conjure up a kazoo to herald the return of Lab Fab? My last post was nearly two years ago, not long after the covid curtains closed. I stopped lab-fabbing because I stopped doing experiments. Over the long interval, I had writing projects. I wrote a paper that presented work I started during my sabbatical and featured many times on Lab Fab (eg., here and here). Hey! The paper got published and if you want a good laugh at the things academics get up to when they escape campus, check it out. I wrote grant proposals, some of which even got funded. Then there were data: image after image to push through analyzing software; extract tons of numbers; and then, multiply, divide, add, and subtract. And of course courses to teach. A professor’s life.

            Well, I thought about blogging the data analysis or even the paper writing, those are after all science-y things that I was doing. But no, what was I going to say: I cut cells C10 through K50 and pasted them into J11 through R51? I gave up. 

            But last month, huzza wuzza, I returned to the lab. I returned to cut paraffin sections of corn roots. A paraffin section is a thing I had not cut since undergraduate days. Fortunately,  I did not have to start from scratch. The lab next door cuts paraffin sections routinely and is all set up. I sought out one of the sectioneers, a postdoc named Joe Gallagher, and asked him for help. He gave me a protocol and graciously allowed me to share some of their solutions and supplies. Now I needed to get started. 

            The protocol has two halves: embedding and sectioning. The embedding part is familiar. I have often embedded samples in plastic and the steps are similar. I sprouted two dozen maize kernels in a black plastic box, with wet paper on the bottom. When the freshly emerged roots were out and exploring, I cut off about a 1 cm tip segment from eight roots and dropped them in a few mL of paraformaldehyde. This step is called fixation, where the meaning of fix is not repair but hold tight, an expedient to keep the structure reasonably intact during the subsequent steps. The few mL of fixative, with roots, are in a small vial, resting on a table that circles, sloshing the solution around to aid the fixative getting into the root tips. A few hours later, I exchanged the fixative for a simple salt solution, and then exchanged that for 50% ethanol. And then every 30 min or so I exchanged one ethanol solution for another more concentrated solution until I reached 100%. This part is called dehydration because the water in the root gets replaced by ethanol.

            Exchanging solutions was delightfully easy because I am used to dehydrating the roots of lab weed, aka Arabidopsis thaliana, the famous model plant species, and like traditional fashion models, unnaturally thin (Fig. 1). The arabidopsis root is around one tenth of a millimeter wide, about the thickness of the cursor line that is blinking at me here as I type (what shows up in in the figure is actually the root system where many roots coalesce). Visible just, but imagine sticking a pipette into a vial containing those roots and trying to suck up the mL or two of solution while keeping the infinitesimal roots out of the pipette. When I had to that, I ran out of curses and instead designed kluges, involving plugs of agarose or wire loops with plastic flaps, anything to keep the little buggers out of the pipette. But by comparison, maize roots are elephants and exchanging the solution was curse-free. 

            Good, because many more exchanges are needed to take the roots from ethanol to molten paraffin at 60ºC. These steps are called infiltration because the paraffin needs time and coaxing to move everywhere within the tissue — complete infiltration. Perched on a chair in front of the small oven set at 60ºC, I worked quickly to exchange solutions without spilling hot wax or letting things cool enough to solidify. Getting a slug of solidified paraffin inside the pipette definitely impedes progress. 

            Finally, comes the embedding itself. Here, I took small plastic weigh-boats, around 1 inch x 1 inch, filled them with fresh liquid paraffin, put two roots in each boat, carefully aligning them to be parallel to each other and to the edge of the boat. By resting the boats on a slide warmer, I had time. Then, I carefully moved the boats off the heat and from there into the fridge, where the paraffin turns to the familiar solid white wax (Fig. 2). 

            Now comes the sectioning. The embedding went like making scrambled eggs, nothing more untoward than a little bit of egg white dribbling down the side of the pan. But paraffin sections are notoriously fussy to cut: the humidity, the knife, the proverbial moon. Still, Joe showed me how he does it, and happily their lab has a tidy work-station set up with the necessary bits. Being nervous, I decided to try my luck on a weekend. Amazingly I got sections. For my purpose, I don’t need many and they came off the knife easily enough. I won’t say easily, I had to jab a small brush at them to stop them curling up in to a tight roll and awkwardly move them over to the slide. I lost a few. But I got plenty. Sections collected and set on the slide warmer to dry down overnight.

            Purpose, I wrote above. What purpose? I have written nothing to this point about why I am slicing up roots in paraffin. That is a story, but it is another story, to be saved for another day. I will also relate how plan-A for these sections failed, spectacularly. Suffice to say that returning to the lab warmed up all sorts of inner spaces. Are things returning to normal? Who knows but the lab is fine vantage point from which to watch.