Kubke Lab:/Notebook/Cranial nerve development/2010/11/24

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Satya Tutorial Day 1

9.30 a.m. - 11.30 a.m.: Firstly we discussed the different approaches for moulding the embryos. There were two incisions made to MH001.

(Note: is it MH001 or RC001? MH001 links are linked to the RC001 page, any reason for this?--MF Kubke 03:55, 3 February 2011 (EST)) The reason behind doing this was to isolate the hindbrain region. During a later tutorial the decision was made not to separate the hindbrain and the head region, instead to make a single incision to separate the embryo at the level of the wind bud. RC001 was moulded whole. Satya demonstrated the technique to create a mould for the tissue of interest using 'Tissue-Tek' OCT. Once the mould was created, the tissue was frozen for 5 minutes at -23 degrees Celsius. We created three moulds overall, one mould with a whole ST22 embryo (RC001). The second and third mould comprised of the ST28 embryo (MH001) with isolated sections of the head and hindbrain. Then we were briefed on the protocol and safety requirements associated with Cryostat cutting. Reuben then had practise in cutting his tissue using the cryostat and mounting the sections to slides. Malisha observed the cutting and helped with the labelling and mounting of slices onto slides. Once our histology tutorial is complete we hope to create a instructional page associated with cryostat specimen preparation and cutting.


(Note: Can you please provide the embryo number codes for the embryos above?--MF Kubke 04:20, 20 January 2011 (EST))

11.30 a.m. onwards: Completing benchmarks set by Fabiana.

Cryostat protocol (Work in progress)

Moulding

  • A square plastic mould was used to prepare the mould which will be used during the cryostat exercise. The mould was coated with a small amount of OCT and the embryo was placed onto the mould in the preferred orientation (flat on). More OCT was added to fill the mould and left to freeze at -23 Degrees Celsius for 5 minutes.

Tip: The best way to get the embryo flat on the mould is by having a coat of OCT on the mould before placing the embryo.

  • Once frozen, the specimen is placed vertically on a "cryostat chuck" so that when the chuck is inserted into the cryostat the orientation of the sections produced is coronal. More OCT is added progressively upwards from the base of the mould in order to provide stability. --Malisha Hettiarachchi 21:15, 30 November 2010 (EST)

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