Kubke Lab:/Notebook/Cranial nerve development/2011/01/19

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Cranial Nerve Development Main project page
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General Entries

  • 9:00 am Lab meeting/journal club. Room 502-B51. Check your email to get the papers we are discussing.

Personal Entries

Malisha

  • 9:00 AM : Lab meeting
  • 11.00 AM: Made an incision at the wind-bud level removing the lower half of the embryo MH006 . Placed the embryo in a larger mould due to previous issues with the eye protruding out of the mould. The embryo was placed in the cryostat( temp -17/-17).
(Note: what is the embryo number?--MF Kubke 06:02, 20 January 2011 (EST)) ( I have added the embryo name and I will be populating the page today --Malisha Hettiarachchi 15:03, 23 January 2011 (EST).)
(Note: Any reason why the page is still empty?--MF Kubke 05:06, 3 February 2011 (EST))
  • 11.50 AM: The eye was still protruding indicating the mould not being deep enough. Added more OCT and allowed for incubation.
  • 12.05PM: Placed mould onto chuck and inserted it into the cryostat. Will allow for approximately 30 minutes incubation time. --Malisha Hettiarachchi 18:13, 18 January 2011 (EST)
(Note: Thanks for pointing out the issues with the depth of the molds. I will look into it --MF Kubke 18:15, 18 January 2011 (EST))

1.00 PM onwards: Began cutting from the head region towards the wing. Slide 1-2 the temperature fluctuated from -16/-16 to -16/-16. The sections were 20 microns thick. The tissue did not look intact and very discontinouos --Malisha Hettiarachchi 21:33, 18 January 2011 (EST).

  • Slide 3: The thickness of the sections was increased from 20 microns to 40 microns , still the tissue looked the same as previously. I increased the " waiting time" before mounting from 45 seconds to a minimum of 1-2 minutes and it improved the sections but it is not very consistent.
  • Slide 6-9 : Focused on mounting the sections very slowly and it seemed to be more consistent . The tissue still does have holes.

Plan for tomorrow:

  1. Nissl stain MH006 sections to determine if the level of subbing is adequate. Ask Felipe if resubbing is an option following protocol A ( cold).
  2. Find a new box of glazed slides to sub with gelatin.
  3. Determine if cryoprotection is a factor in the discontinuity of the tissue.
  4. Attempt to dissect an embryo
  5. Histology

--Malisha Hettiarachchi 04:08, 19 January 2011 (EST)

Reuben

Nissl staining of sections cut yesterday. (RC009)

  • 10:30am-12:30pm Stained odd numbered slides.Coverslipped immediately afterwards.
  • 1:00pm-2:15pm Stained even numbered slides.Coverslipped immediately afterwards.

Organized the lab also during morning incubation times.

Sections didn't fall of the slides indicating that the subbing was successful

  • 2:45pm Working on my online entries. --Reuben Cutfield 20:47, 18 January 2011 (EST)

Fabiana

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