Kubke Lab:/Notebook/Cranial nerve development/2011/01/18

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General Entries

  • I have now added a full version of the gelatin subbing protocol (thanks Reuben for getting it started and Malisha for helping me write it). Felipe says I need to change a couple of things so I will do that tomorrow. Kubke_Lab:Protocols/Gelatin_Subbing
  • I also added a field on the embryo log page - Malisha noticed that the template form did not ask whether the embryo was cryoprotected or not, so I added that to the template. Please make sure that field is in all of your embyro entries
(Note: Another field (labelling and storage) has now also been added--MF Kubke 05:59, 20 January 2011 (EST))
  • I am in the process of cleaning up the templates to make them easier to use.
  • When stating the embryo stage, please make sure to clarify whether the staging was confirmed by me or not

Personal Entries

Malisha

  • 10:00 AM - 11.00 AM: Cleaned slides stained yesterday using 95% Ethanol and a razor blade.
  • 11.30 AM: 12.15 PM: Observed Fabiana change the Subbing slide protocol.
  • 12.30 PM onwards: Subbed slides using the following protocol (version: Revision as of 12:16, 18 January 2011) any amendments will be mentioned here. --Malisha Hettiarachchi 04:12, 18 January 2011 (EST)
(Note: Mal, I added the version of the protocol that you used for this, Felipe wants me to change a couple of things, and it is important to refer to the specific version that you used --MF Kubke 05:23, 18 January 2011 (EST))

- Okay I will do this from now on.--Malisha Hettiarachchi 15:01, 23 January 2011 (EST)

  • Spoke to Fabiana about the Nissl protocol. She advised that the protocol should be ammended because we are not working with paraffin blocks. The following steps should be used H20--> Crysl Violet-->H20--> 50% Ethanol--> 70/75% Ethanol--> 70% Ethanol with acetic acid( approx 5 drops)-->95% Ethanol-->100% Ethanol 1 --> 100% Ethanol 2--> XYLENE 1,2 and 3. The timing of the Crysl Violet step and differentiation step( 70%eth and acetic acid) should be moderated. --Malisha Hettiarachchi 19:51, 31 January 2011 (EST)
  • Feedback form fabiana for MH005 . The warmer temperature works well however neuroepithelium not keeping together. Cut at thickness of 40 microns and 0 degrees( or slightly above 0 degrees) knife angle.

Reuben

  • 10am-4pm Sectioning of embryo RC009 in the cryostat. Mounted sections onto the slides subbed in gelatin yesterday. Slides were labeled RC009 1-13 and kept in the fume hood overnight to dry. Every single section has had the speed of cutting and time spent in chamber before mounting as well as any observations recorded into my lab-book. As well as a map of where every section may be found on the slides and which sections weren't mounted such that one can easily see whether a particular group of sections are serial or not.
(Note: It looks to me like you stopped cutting before reaching the end of the hindbrain. Is there any reason you did not cut the whole way through the head? --MF Kubke 21:03, 18 January 2011 (EST))

I did not leave myself enough time to complete the sectioning of embryo RC009. Half the hindbrain and head were discarded and were not captured on the slides.--Reuben Cutfield 22:39, 18 January 2011 (EST)

Fabiana

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