Kubke Lab:/Notebook/Cranial nerve development/2014/12/09
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Today I had one successful injection (the stage 19.5!!!) and it was the most beautiful one I have done so far. Very, very clear images of discrete axons, it just had this clarity to it that I haven't had before. The stage 24 embryo (HK022) where I injected the basal plate didn't work because I didn't inject deep enough past the connective tissue, so it dyed the connective tissue well but when I cleared that there was no dye on the actual hindbrain left. I am currently waiting for Fabiana to get back from her meeting because I can't wait to show her my embryo. While waiting for the glycerol to clear the embryo, I dissected and injected a stage 18 embryo with attempts to replicate what I had done on the emnbryo that i stained so successfully. I realise that I need to improve my basal plate injection technique so I will play around with that tomorrow. In my stage 19.5 embryo that I mounted today, I dyed the trigeminals with DiO and the VII/VIIIth with DiI and with the green filter, I saw some interactions between the two, because the two colours show up together on it. I will have to talk to Fabiana about what they are though because from what I think it looks like, it seems as though there is an afferent from the Trigeminal going into the 8th nerve? crazy and I'm sure it's not true but I've taken the same photo through different filters and so will combine them on photoshop to see if I've imaged the same axon twice or what the deal is.
I just learnt how to blend images of different layers of focus on photoshop which is great and going to be so helpful and it is incredibly easy! Very happy about that. I imaged my HK023 embryo (my stage 19.5 that I love) on the Nikon this afternoon and I know Fabiana would probably have preferred that I have supervision for that but I was extra careful and followed my instructions carefully when turning things on (and off) I just knew I had to image these on a higher quality microscope because I think that this was a really good embryo for data.