schto eto za research?

so i've never really fully described what i'm doing for research, so here goes... i work under mike redd who is the faculty professor in the Trede lab at hunstman. im his only undergrad, which is nice, because he's pretty busy. but he let's me do all my own experiments and keeps me on a pretty loose leash. he basically "trained" me by showing me everything once, and i just tried to take good notes and ask around if i had any questions. but i've got it all down pretty well now. this may be a little boring but i want to lay out the whole procedure in detail, just for my own memory.

Thursdays: i set up fish, by separating out mating pairs into separate breeding tanks, and letting them love each other overnight.

Fridays: I take the fish down, by pouring the fish back into their main tank and then pouring any embryos into small petrie dishes for cleaning and sorting. the embryos are kept separate from the fish in the tanks because they fall through a perforated inner tank and rest on the bottom where the fish can't get at them, because they will eat them. after the eggs are all harvested (there are usually hundreds), about 100-120 are separated out and rinsed, getting rid of any unfertilized or rotting embryos, and any that might be showing signs of abnormal development.

Mondays: after the embryos have been sitting in an incubator over the weekend, they are now little swimmers with eyes, a pumping heart, immune system, and circulatory system. it's crazy how quickly they develop. if you've got 15 minutes, you can actually watch cell division. it will change your life. at this point, i soak the fish in a certain drug that we are using and let them swim around a little in it for like an hour, and then i make a small wound in the back part of the feathery part of their fin, which begins an inflammatory response. i let them sit in the drug with these wounds for a couple of hours and then fix them in a formaldehyde solution that crosslinks all of the cellular components to freeze them all at that moment in time.

Tuesdays: i wash the embryos 3 times for 5 minutes and soak them in a block solution to prepare them for their first antibody. this antibody binds to certain cellular components and will give a place for a second antibody to bind the next day.

Wednesdays: the embryos are washed 4 times for 20 minutes each time, and then soaked in a second antibody that has a pink fluorescent tag on it that will allow all of the leukocytes to show up under fluorescent microscopy.

Thursdays: fish are setup for the next week, and the fish from the current week are washed 4x20 and then run through a glycerol series of 30% glycerol, 50%, and 80% and left in the 80% overnight.

Fridays: fish are taken down, and the current fish get their heads separated from their tails, and the tails are arranged on a microscope slide, and then examined under a fluorescent microscope. under one filter, you can see all of the leukocytes (white blood cells) and i count them. under a different filter i see only neutrophils, which are a specific white blood cell that has been genetically altered to carry a gene for a fluorescent green protein called GFP. these glow green under the different filter, so i can differentiate how many of the total leukocytes are neutrophils. i can then compare counts with the control slide, to the counts from the slides from fish at different drug concentrations, and see if the drugs have any sort of effects in the inflammatory response.

as i've been doing this process i started getting frustrated with the lack of accuracy and repeatability with the experiments and decided i would start messing around with it and testing different things. so i started making things really sterile with some of the fish, bleaching them and putting them in filtered water. some of the other fish, i soaked in bacteria rich water, made from rotting fish embryos. the surprising results are that the bacteria water, drastically increased neutrophil counts, while reducing macrophage counts. this was something no one expected, and i'm doing other tests to fine tune things. but to make a long story short (or not as long), the lab is potentially going to redo how they perform their experiments to reflect these findings. everyone is all excited about it, so it's pretty exciting, but now it puts a lot of extra pressure on me to get this figured out. crazy, but i'm loving research.