I need to dissect fresh pituitary glands from mice and use them for protein AND RNA extraction. I tried to follow a video published on JOVE but was not an easy job as shown and I totally destroyed the brain and could not see the gland
Hi Bassem,
I don't know how it was shown in that video, but we use a very simple technique, which only requires a scalpel and a steady hand, yet you may need some practice nonetheless. :)
When the brain is removed, and you see the pituitary between the two strands of trigeminus nerves, you carefully detach it (ie cut through the dura mater with the scalpel)on the left and right side (where it meets the nerve strands). Then you carefully slip the tip of the scalpel underneath the gland and lift it out. That's all. :)
Good luck! Christiane
Dear Bassem,
I have done it in the rat and mice. It is very simple. Just dissect the dorsal cranial bones, and after exposing the entire dorsal surface of the brain. Make an incision to at the rostral end (caudal end of olfactory bulbs) and caudal end (mid-cerebellum). Hold the brain upside-down, with it is weight the brain tissue usually easily comes off with gravity, if not give it a bit of a assistance with coarse forceps. Once the brain drops off, then you will see the pituitary sitting inside the bone or fossa (cella turcica). Then you just have to get it out with forceps, pay attention if you need to keep tissue integrity for histological purposes.
If you can get hold of Prof Gary Housley at UNSW, he knows it better than I do:-)
best wishes,
Refik
I will try to contact Gary at UNSW and hopefully he can help me out
Hi Bassem,
If you can follow the instructions that I have given it is very simple and you wont need help. Which department are you in? I thought you were working in the same department as Gary in the Translational Neuroscience Institute. OK I just checked and you are Anatomy. Who is you supervisor?
best wishes, Refik
Can you see the pituitary once you dissect the brain out? Are you working with fixed tissue for histology or fresh tissue for gene analysis?
I want to get fresh tissues for WB. The few times I tried, I managed to lift up the brain but failed to see the pituitary that makes me believe that i move it with my rough technique of lifting the brain up
OK it is harder to get it out when the tissue is unfixed and with bleeding etc. Just make sure you have lots of PBS in a beaker and wash the blood away with a pasteur pipette to increase the visibility. It is easy to make a mistake when you can't see anything. Also once you remove the brain, use a stereomicroscope to look into the area (cella turcica) where the pituitary should be sitting. It is a small tissue but big enough to see with naked eye, however having stereomicroscope will help at least at the start. Once you learn you will do it your eyes shut:-) I am not sure if I have explained the method in my PhD thesis but it is available at RG, you should check it out anyways for detailed methodology. I will actually insert it for you. Be patient, it is over 500 pages and takes a little while longer than average pdf file to download:-)
Best wishes, Refik
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I did it... followed your advice and worked very well and easy... But came out all broken,... perfect for protein lysates,,, but will be very challenging if I want to have a look on their histology... Do you think I will have to perfuse it or may be with practice I will be able to dissect it intact from fresh tissue? Thanks again for your help
Or may be I will drop the Brain and then fix the remaining part. This should make it easier for me to dissect the intact gland for histology