Kubke Lab:/Notebook/Cranial nerve development/2010/12/17

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General Entries

  • Malisha on leave - will return after the summer break towards the end of the first week of January.--MF Kubke 17:18, 16 December 2010 (EST)
  • Asked Reuben to give the new crysotat knife angle a go--MF Kubke 17:18, 16 December 2010 (EST)

Personal Entries

Malisha

On Leave --MF Kubke 17:17, 16 December 2010 (EST)

Reuben

Cryostat Sectioning of chick embryo RC004

  • 11:00am Staged Embryo RC004 at Stage ST25 under a dissection microscope.
  • 11:15am Embryo cut saggitallycoronally immediately caudal to the wing bud.
(Note: Do you mean coronally?--MF Kubke 05:08, 20 January 2011 (EST))
  • 11:30am Loaded Embryo into a plastic mould. Incubated it in the cryostat chamber at -17 degrees C.
  • 12:00pm Placed the mould onto the metallic chuck. Incubated at -17 degrees C in the cryostat chamber in the chuck holder.
  • 12:30pm Began cutting.

Following a discussion with Fabiana a slide-map was created in my lab notebook whereby each section loaded onto the slide was given a letter and the details regarding each section were listed alphabetically whilst the placement of each alphabetically labeled section was diagrammatically represented in my lab book. In this way I can quickly section and mount with different parameters and an observation of the section quality should reveal which set-up works best.

  • 1:30pm Changed cryostat object and chamber temperature to -19 degrees C and added more OCT to the rear of the mould.
  • 2:15pm Resumed cutting at -19 degrees, 5 degrees angle to the blade, 25 microns thick.

Made multiple sections varying the angle of the specimen to the blade, the thickness of the section, the speed of cutting and other recorded variations.

  • 3:30pm Stopped sectioning. Cleaned up machine and placed the remaining tissue into the freezer.
(Note: Why was the rest of the tissue put into the freezer after we agreed we shouldnt do that? What happened to the tissue?--MF Kubke 04:50, 3 February 2011 (EST))
(Note: We can't leave the blocks in the cryostat because it is set to an automatic defrost so it was either discard the tissue then or put it in the freezer until we next needed the chuck. The tissue was discarded the next time we cut.Should we be keeping them?)
(Note: Did you cut the tissue after putting it in the freezer or just discard it? I am confused, if the tissue is to be discarded why is it put in the freezer? trying to understand what you did. --MF Kubke 22:46, 3 February 2011 (EST))
(Note: The tissue was placed in the freezer after cutting it. It made sense to put it in the freezer at least until we needed the chuck.)
(Note: Did you cut the tissue after putting it in the freezer or just discard it?--MF Kubke 17:00, 6 February 2011 (EST))
(Note: Discarded it. Was this the wrong thing to do?)

Fabiana


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