Monday 5 September 2011

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

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