Toaster the Psychic Organ Builder

So the other day I was standing inside of a 4-story pneumatic pipe organ while it was being played thinking to myself: "Holy crap I'm inside of a 4-story pneumatic pipe organ while it is being played!" when it occurred to me that maybe I might actually know something. For a while I've been feeling like I have spent so much time and effort learning about molecular biology that I no longer know anything actually useful outside of the laboratory. This feeling was jade creeping into the awesomeness of science, and I found it best to promptly shake it off like a dog after a bath and go roll around on the floor joyously with all my legs in the air. I mean, sure this science sometimes appears to be nothing more than moving tiny amounts of expensive liquids around while muttering vague incantations about hypotheses and replicability. But what it is, what molecular biology fundamental is, is me dissecting LIFE ITSELF with my MIND!!! I could have all the fancy tools an R01 can buy at my disposal and a hammock above my bench, but those things wouldn't mean crap for meaningful, insightful science if I didn't start by designing an elegant, productive experiment. It's like I'm telekinetic at the molecular level, because I have to imagine the true nature of all these stochastically interacting proteins, genetic elements, or other molecules and their aggregate behavior clearly, I have to fix it in my mind and strip away the limitations of my vision to harness the quiet power of chemical reactions and hydrogen dipoles to prove the accuracy or folly of what I have captured with my mind.

Sure, this science doesn't have the immediate satisfaction of swinging a hammer and building a chair, the delay to sate the fundamental human need to create and explore burns on a longer fuse, but oh, when it connects it is all the more beautiful. I may not be building a pipe organ, but I am reverse-engineering something many more times intricate when I can't even see it with my naked eyes. That. Is. Awesome.

Marathon Science

My boss told me that I should factor in that everything will take much longer when I have many samples and that I should plan my time accordingly.

She was very right.

In fact, she was so correct that I wound up pulling 29h in the lab, and 14h the day before, to get Experiment done. There weren't that many samples to begin with, but by the time that I split those samples into cell culture under different conditions and then stained each of those conditions for different sets, I had a couple hundred samples to run on my hands (or, rather, in a tray because I couldn't carry them all at once).

What I found most interesting about this (aside from the Science of it that I can't blog yet), was how all-night science affected me. The entire night was an odd rollercoaster of exuberancy and despondency.

11a - Got to lab, felt guilty about being late but vindicated by 14h there yesterday.
12p - Relieved to see that samples had not drowned in melting ice bath overnight, put into refrigerator to finish staining later because Machine was scheduled out into early evening.
1p - Indian food buffet!
2p - Talk to visiting scientist, then journal club.
3p - Journal club.
4p - Realized that I didn't have enough Enzyme, so I went to go get more from our super-convenient on-site Enzyme Store.
5p - Started finishing staining.
8p - Finished finishing staining, Machine time.
10p - Done with Machine for now.
11p - Pulling cell culture.
12p - Plating for staining, blocking, singing deliberately off-key.
1a - Sandwich and caffeine time.
2a - Primary stains, dancing around laboratory to punk music.
3a - Washes. Laboratory is very cold. Sweater not helping. Note that this parallels drop in body temperature that occurs when sleeping. Turn up lab thermostat just a little bit. At least it's warmer in the cell culture room.
4a - Fixation. I could have gone home and slept at this point, but didn't want to have to come back in later. Arikia and I discuss what we carry around in our backpacks/bags, she rightly points out that the contents of mine would have me on a terror watch list in no time flat. I have my laptop, a zombie comic book anthology, lots of heavily annotated biology papers, a screw driver with a full tip set (even hex wrenches!), a digital multimeter, wire strippers, a soldering gun, needle-nose pliers, gloves, fisticuffs, a gigantic rubber band, a set of Exacto knife scalpels, some faded index cards, cufflinks, a bunch of batteries, and Pepto Bismol. I do not know how old that Pepto Bismol is. Might need to add a crowbar.
5a - My pellets have disappeared. I have become paranoid and cynically convinced that all I'm doing is spending lots of time moving small bits of liquid around even though I know from doing this before that fixation can cause a change in their color.
6a - I found enough change in my pockets for a vending machine Snickers bar. It's dawn and I am shivering even with my sweater on.
7a - Still washing and enzyming, other people start to filter back in (this is Saturday now, right?).
8a - More washes and a brief 30min nap on the couch in the departmental library, which is surprisingly comfortable. Glad my phone can be used as an alarm clock, also glad that protocol has 40min incubation periods.
9a - Still can't see any pellets. Morose about prospects for data. Still washing.
10a - Final stain!!! Just one more wash...the sound that the suction makes when washing no longer amuses me...
11a - Machine time! Crap, gotta restart the machine. Fairly certain that I dozed off waiting for the machine to start up all its pumps and stuff, woke back up when I started to fall out of chair.
12p - Staining controls calibrated.
1p - Running samples, strangely extremely alert, become completely attuned to Machine such that I am already fixing a problem before it pops up a problem-prompt.
4p - Finished running samples, Machine no longer talking to me.
4.5p - Promptly miss only bus home for next hour, decide to walk instead.
5p - I am lost. I know by dead reckoning exactly where my apartment is, but there are all these inconvenient streets and houses in the way.
5.5p - Home.

Truth be told, I still didn't go to sleep until 1a. First I ate a lot of food, drank a lot of drink, and played with the new power tool that finally came in the mail even though I didn't have anything on hand that expressly needed it. I need to find space in my backpack to add this tool. And to top it all off, I even had trouble falling asleep.

UPDATE - Dremel is now in my backpack. Also found bag of 15 2" 30-Ohm speakers in there.

Actin Belly

40X coronal stomach section, stained for actin. Click for high res. Scale bar = 200um.

Frozen section, wash off excess OCT in RT dH2O, 10min permeabilization in 100% acetone, 30min block in Toaster's Übermegablocking Buffer*, added primary polyclonal Ab without washing, incubate 1h @ RT, wash 3X in Washy Wash Buffer**, add labeled actin at 1:200 and secondary labeled Ab against primary at 1:10, incubate 1h @ RT, wash 3X in Washy Wash Buffer, tap off all excess wet, 2 drops Prolong Gold Antifade + DAPI (@RT), coverslip w/out bubbles, cure O/N in a dark box.

The resolution I managed to get here is so exquisite and the result so beautiful that I am very tempted to get a 5'x6' giclee print of it and hang it on my everything.

*Toaster's Megablocking Buffer: 1% BSA, 1% FBS, 1% NGS in 1X PBS; to make Toaster's Übermegablocking Buffer add 1:10 CD16/CD32 to block endogenous Fc antibody receptors in the tissue.
**Washy Wash Buffer: 2% FBS, 0.02% NaN2 in 1X PBS

Proof

In case any of you doubt the awesome hotness of the science I have done this week, gaze upon the picture above and be unimpressed (taken today on an Olympus upright fluorescent microscope). Then click on it to kick it out into a new tab or window much bigger and you'll see just how sweaty-palmed hot this science is.

Don't see it?

Look to the resolution.

See?

OHHH YEEEEEEAH!

This was done using special capillary slides and a Microprobe apparatus and an abbreviated incubation timeschedule. Despite my custom Megablocking Buffer working wonderfully in other micrograph fields, this particular chunk had particularly high background so it's scientifically useless. Still beautiful, though.

Post Script - Toaster has finally gotten a toaster. I've been without a toaster for 8 months, and I've been quite aware of the irony of me, Toaster, not actually having a toaster and being forced to make toast with a skillet or the oven. But this one has 4 slots, and each one is big enough for a grilled cheese!