Kubke Lab:/Notebook/Cranial nerve development/2011/02/03

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Malisha

  • 10.00 AM: Opened a google doc to plan studentship report
  • 10.30 AM: Working on neuroanatomy document. Goal is to complete the necessary components by tomorrow--Malisha Hettiarachchi 17:04, 2 February 2011 (EST).

Reuben

  • 10:00am Photographed embryo MH007
    • Severed embryo rostral to wing bud and re-photgraphed.
    • Placed embryo in large metallic mould half-filled with OCT.
    • Incubated mould in the cryostat at -19°C.
(Note: The large mould took 45 minutes to freeze and harden.)
    • Loaded the mouldblock onto a metallic chuck and secured it in the metallic chuck holder.
(Note: not sure I understand - What is the difference between the mould and the old plastic mold? I assume you took the block off the mould before mounting it? --MF Kubke 03:48, 3 February 2011 (EST))
(Note: Can you please clarify, what is the difference between this metallic mold and the old plastic mould? What prompted this change?--MF Kubke 22:52, 3 February 2011 (EST))
(Note: Malisha was always complaining that when she embedded the later stage embryos that the embryo would protrude out the side of the mould. You told Malisha that you would look into getting larger moulds. Since I was cutting a ST25 embryo I sought to find and use a larger mould. I will be uploading pictures of it so you can see it.)
    • Built up OCT slowly to support the large block.
(Note: Adding OCT too quickly causes the frozen OCT to melt.)
    • Using a razor blade, by hand, I trimmed the block into a cone shape and removed enough OCT so that the block could fit between the chuck holder and the knife.
  • 12:00pm Began incubating the final assembly at -19°C in the cryostat.
    • Went to lunch.
    • Entered in todays journal entry and added page for populated the MH007 page with the appropriate templates.--Reuben Cutfield 21:02, 2 February 2011 (EST)
(Note: You didnt add the page for MH007, Fabiana did. --Malisha Hettiarachchi 18:58, 2 February 2011 (EST).)
(Note: Due to two main factors I can identify the cutting of MH007 is both taking a long time and producing poor tissue quality. The first reason is that I used the end of a bottle of OCT. Squeezing the OCT out produced a lot of bubbles which I found difficult to remove once it had hardened. There were many sections lost following the first slide. I had to cut out one of the main bubbles and refill the hole with OCT and wait for it to harden. Secondly Satya's Histology Lab is immensely hot and humid. For some reason it appears to be far hotter than even the hallway next to it. The chamber is warming up considerably and often reaching -16°C. At -17°C The sections begin to stick to the glass cover and it makes it very difficult to mount. The humidity is causing a lot of moisture build up which is both crinkling the OCT and warming the sections adding to the sticking. I will continue to cut the rest of the embryo but am concerned with the method/conditions.)
(Note: what do you mean by the 'histology lab upstairs'? --MF Kubke 03:48, 3 February 2011 (EST))
(Note: Can you please clarify, did you cut in Satya's histology lab?--MF Kubke 22:51, 3 February 2011 (EST))
(Note: Have clarified. I will refer to the lab as 'Satya's Histology Lab'. Sorry for the ambiguity.)
(Note: Another factor could be the stage of the embryo? This is a stage 25 and we have had trouble with older embryos up till now.--Malisha Hettiarachchi 15:36, 3 February 2011 (EST))
(Note: I dont think so Mal, older embryos should be easier to cut - but who knows --MF Kubke 17:02, 3 February 2011 (EST))
  • 4:10pm Finished cutting MH0007. Will stain the slides tomorrow. --Reuben Cutfield 22:14, 2 February 2011 (EST)

Fabiana

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