some lab photos

me in the confocal attempting live imaging!!!!!!!

last post.......

Last week I finished my placement in the University, during the last week I attempted to do another in situ hybridisation in order to identify where my gene of interest was expressed. Although we managed to get staining I think we rinsed the sample very fast and the majority of the staining washed away making the slide really faint and hard to visualize under the microscope. Unfortunately it is  a very tricky technique that you need to optimise for your own probe and mRNA of interest. The protocol states that the staining can be left between 10 minutes and 24 hours so, mmm hard to get it right. On my first attempt my friend and I allowed to stain for about 2 hours and the majority of our sample was really over stained and this time round we allowed the reaction to proceed for about an hour and 15 minutes and it was under stained!!!!! Shame I feel like we were so close to get it right but unfortunately my time in the lab has finished and now I got to prepare for my third year that starts next Monday!!!!

Last year I read a couple of comments before deciding to do a summer placement and now after having done it the only thing I can say is that if you are considering to do a summer placement and you are reading this.... go for it!!!! It is well worth it and I have learnt a lot, I feel more confident in the lab and more independent I feel like now I am ready for my third year project and possibly a PhD xxxx

Adriana

last week....

Last week was very exciting because I did an in situ hybridization, something I had not done before in practicals. I did it following a protocol in a journal.
The purpose of the in situ is to localize the expression of the gene that I am interested in as well as getting some idea of the level of expression but since I had never done that before this first attempt was really just to get hold of the technique so we only did two different phenotypes and a control. We already knew where both genes were supposed to be expressed and our control just did not have a probe.
For the two genes one was slbo which is inlvoved in the signalling pathway of differentiation of border cells so I was expecting to see the antibody binding in border cells and that actually happened so im thrilled because of that. Our second gene was domeless and that one did not work very well.
We also experienced some problems with the control which was really disappointing !!!! Since my control only had hybridization buffer I was not expecting to see any antibody binding at all and I was expecting some clear ovaries but unfortunately I got some binding in the cytoplasm which makes us think that the antibody is not specific enough!!!!and is clearly binding to something else.
Before I arrived another student doing a summer placement tried to perform an in situ and he had a lot of background staining and non specific binding so my supervisor order some fresh antibody to rule that possibility out, unfortunately even with the fresh and preabsorbed antibody there was some binding.
I will have to talk to my supervisor about this issue but I might also try increasing the concetration of the probe and decreasing the staining time to try to get only very specific binding !!! I dont really know where to go from here so as soon as I know ill get to it.
I have also continued preparing some more slides everyday and I cna say my dissecting skills are really improving =) plus my slides look much better!!!!
xxxx
adriana

today..................

Today I had a great day!!!!  I shadowed lovely Lauren, she is a PhD student in our research group and she works in live imaging. I knew live imaging was part of my proposal and I knew it was something I would have to do in my summer project,  but I didn’t give it a lot of thought and didn’t really realize what it was all about. I can say this is going to sound terribly stupid but I did not know that you image the cells when they are still alive!!!!!! I know I should have gotten a clue by the name live imaging hahha but I really didn’t. I think I just didn’t think about it much, and I thought that live imaging was just a lot of images together taken from different slides and then made a movie hahhah sort of what it really is but I cannot believe is the same egg chamber that you are looking at and that all of those pictures come from the same border cell migrating towards the oocyte I can say that today I  actually saw live cells moving!!!

As soon as I came into the lab I was assigned to work with Lauren and she explained me how to prepare the media for the cells, she showed me what you had to add to the media so that the cells think they are still in their cosy natural environment!!!! And so that they stay alive!!!! She allowed me to help her adding the aminoacids and antibiotics and she let me do things on my own with her supervision which was brilliant nerve racking and challenging!!!!!  We first prepared the media and then went to the prep room to adjust the pH to 7 which she also allowed me to do myself !!!  in the prep room something really cool happened as well, there was another PhD student working there  with liquid nitrogen. I have read the words liquid nitrogen many times (x ray crystallography lectures........) but I had never seen actual liquid nitrogen it was amazing!!!!!!!!!

 Never mind back to the topic having prepared the media  we went into the fly lab to dissect the ovaries which has to be done extremely carefully so that they don’t die, and afterwards we headed to the confocal microscope to do the live imaging. I had worked a bit with this microscope before but doing it in real time is so different and just amazing to me!!!! I know that’s as geeky as you can get but I’m not going to lie I was thrilled!!  I just really wanted those border cells to move!!!!!!

With live imaging you can actually spot when and where a defect in migration takes place but for today we stuck with wild type cells to visualize normal migration patterns first.

 I really enjoyed my day plus I learnt a lot about how the microscope works, how to set it up, how to use it wow loved it!!!!

Xxxx

lately.........

I haven’t written in a while and the reason for that is that I have continued doing the same thing. I am still dissecting ovaries of drosophila flies that have in them altered genes either kinases or phosphatases in the hope of finding a mutated gene that will have an effect on border cell migration that we can investigate further and relate to metastatic ovarian cancer.

I suppose that working so intensively in only one project has made me realize the reality of the amount of time that research entails. It is not easy and it takes a long time to get results!!!! We still have a lot of flies to screen before we finish and I hope we get some results especially because the people in my lab work really hard and I hope it pays off!!!!

In the following weeks I will try to perform an in situ hybridisation and also shadow a phd student that is working on live imaging hoping that I can grasp the procedure and have a go at it by myself. Unfortunately it has taken her a long time to master the way of doing it so it does not sound easy at all!!!!!! As soon as I embark in a new activity or get some interesting results I’ll post something up!!!! I have also enjoyed thoroughly being part of the Friday meeting where one person in the research group talks about their resent work I think I have learnt a lot and I sort of thought that they were all working on exactly the same thing. Now I have realized that they all approach the topic in quite different ways and there is so much to learn out of each person involved. So far I feel really happy and I am over the moon of having the opportunity to see how real research takes place

Xxxx

Adriana

last three days

the last three days I have still been working on dissecting ovaries from drosophila flies and them preparing the slides to visualize them but now I understand a bit more the reason why we are doing this, I say we cause we are in fact about 6 people all doing the same. We set up crosses of flies in order to obtain progeny with all the characteristics that we need. one of the flies has a slbo that as i mentioned before makes it border cell specific and this is linked to gal 4 gal 4 in turn is used to switch on the expression of gfp by UAS and also works to swicch on the dsred that is a red dye with a nuclear localization signal overall the genes in that chromosome help us identify and visualize the cells we are interested in but also we need to make sure they keep these genes so recombination is avoided by having a balacer chromosome. which is a chromosome that has a lot of inversions and avoids recombinations, I have been informed that these fly lines with balancer chromosomes were created by radiation. The balancer chromosome also has another gene that helps us identify what we are looking for and that is it has the cyo gene that makes the wings of a fly curly ( I'll come back to that now). Now the cross also has two chromosomes in one it has the UAS transgene and in the other the cyo. The UAS transgene is what we are interested in because it is the one that we have manipulated to either overexpress or inhibit a kinase or a phosphatase enzyme, the other is used to screen the flies, that is if the flies have curly wings they have inherited the chromosome that we are not interested in whereas if they have straight wings they have the UAS transgene.
What these means is that we have to screen these flies and there are so many of them, they are all labeled with the different kinase or phosphatase that has been altered and when we dissect the ovaries and fix them into a slide it is then possible to screen the slides looking for alterations in migration and movement. The past three days I have been improving my dissecting technique as well as my fixing technique. I really struggled getting the slides to a good standard with a little background that allows the visualization of the slide but I have definitely improved specially today that I manage to produce really good and clear slides. I suppose the key was to allow the PBS to rinse for a longer time !!!!! I am getting there and I am getting better at it !!!!