With my second experiment, mixed success . . .

I just finished looking at the slides from my second attempt at double immunofluoresence. It worked but not perfectly. Remember I wanted to stain two different proteins? For technical reasons we changed the proteins we targeted. We set out to stain ERa as before but the second protein was GFAP. A marker protein for the non-neuron cells in the brain called the glia. My GFAP staining (red) is beautiful! The type of glia I stained are called astrocytes because they look like stars. The fluor is called Texas red; it’s the color of a little-red-wagon or a classic red tulip. I’m going to learn how to take a picture with the very very fancy microscope they have here so I can show you in the future. This scope is so automated you don’t even change the objective lens by hand; you push a button! It feels a little like I imagine the astronomers at NASA feel with they operate a Mars rover! OK so we celebrate the bright red GFAP staining. Unfortunately, the mellow green (like a watermelon pickle) that should be in the cytoplasm or nuclei of neurons is in the same place as the red! Oops! We had something called cross-reactivity because we didn’t wash the slides separately. The secondary antibody (see my previous entry about the immuno technique) that supposed to latch on to the primary antibody for ERa seems to have connected to the primary antibody for GFAP instead. This is not as stupid a mistake as it sounds we had reason to suppose that we wouldn’t didn’t need to worry but more care – more steps – more time – probably would have yielded more satisfactory results. These have been two good half weeks in the lab. I’ve been working in an area of technique that is similar to one that I’ve practiced before and successfully guided students to perform. But this version is trickier because it is more complicated. More things can go wrong so you need to exercise even greater care to control more variables and eliminate more sources of experimental error. If I had more time I could repeat the experiment again and I think I would get it right this time. The researcher who worked with me and taught me this technique, Debora Rothman, will be continuing with the experiment because it is important to the work of this lab that the presence of ERa in the nuclei of brain neurons be confirmed with this technique. Next week I’ll be moving on to another area of Dr. Shannon Weickert’s lab. I will be working with another member of the lab to culture, transfect (insert pieces of DNA) and study the response of cells to a stimulus using light produced by a firefly gene as the means of measuring response. It’s a really neat technique that I understand in theory. I’m anxious to see how it works in practice. But more about that next time.