Week 2 at my lab started off with just reading at my desk. I would always
get there around 9 but Neha would usually get there at 10:30 so for an
hour and a half I just read papers, checked my emails, and did any
unfinished work from the previous day. The Matsunami lab has a journal
club where all the grad students present a paper they read on Monday and
Hiro buys us lunch. It's like when we presented the scientific papers we
read in fall term only much longer, more sophisticated and of course with a
more in-depth understanding of the topic itself. All these papers are
related to olfaction in one way or another. This week Neha presented a
paper on "Sexually Dimorphic Neurons in the Ventromedial Hypothalamus"
that govern mating in both sexes and aggression in males. I tried reading
the paper in advance to prevent any "getting lost" but eventually she lost
me in the middle of discussing the different techniques. The parts that I
understood were really interesting. Next week, Jianghai is going to present
on Ectopic Olfactory Receptors and I have yet to read the paper!
After the meeting, we did Phospho S6 staining on four slides (2 control and
2 test), Caspase-3 staining on another four slides (2 control and 2 test)
and 2 negative controls. Phospho S6 and Caspase-3 are both antibodies
that will bind to the respective proteins in the cell. The S6 protein is
phosphorylated only when mRNA is being translated so the test mice
(which were put in the two paper buckets with acetophenone) slide should
have more staining than the control mice because acetophenone is
activated. Then we added a secondary antibody that would bind to the
primary antibody (Phospho S6 and Caspase-3). We also washed the slides
with blocking solution (which is actually skim milk!) because it prevents
any non-specific binding of the antibodies. We want our secondary
antibody to bind to our specific primary antibody. With the Caspase-3
staining, the KO mice without the RTP1 and RTP2 proteins would have more
staining because casp-3 is an indicator of cell death. Neha told me that the
smell cells of the mutants die quicker so there would be more staining in
the KO mice than in the WT mice. The 2 negative controls just had the
blocking solution and no antibodies.
After the staining we put the slides in the cold room (-4 degrees). We then
took pictures of the slides on the microscope with different light filters. We
took all the photos in black and white so they're not as pretty as you would
see on the microscope. Yesterday I started counting the positive cells for
the staining, and the number of cells with nuclear staining (bisBenzimie).
Crider came to visit at around 4! and I introduced her to Neha. Neha
showed her around the lab, microscope room, and the dissection lab. She
also got to meet Hiro!
Then Crider took me and Meg out for dinner! The food was good, but I'm
not going to comment on the restaurant itself! Thanks Crider for a nice
evening! You made me think of the bike trip and, oh, how I miss it dearly!
YOBTO '12
The next day, Neha went to the animal house and clipped the mice pups'
toes to collect DNA. Thankfully I didn't have to do any of that. She came
back with 15 tubes each containing little toes - which freaked me out at
first but then we mixed tail buffer and proteinase K to each tube to degrade
the proteins. We put these tubes into the PCR machine. Then we made a
master mix that contained water, 10x buffer, 2mM dNTP, Taq polymerase,
and the forward and reverse primers of the sequence. We centrifuged the
tubes and ran gels to determine the genotypes of the pups.
On Friday, Neha transferred the pictures we took from the microscope to
her computer and I spent 4-5 hours counting the number of cells in the
neuronal layer, the number of positive cells from staining and recorded the
area of the neuronal layer. Although I don't like the computer work very
much, my work in the lab has been very balanced between hands on
experiments and data analysis on the computer.
Hope everyone else is having a good time!
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