Kubke Lab:/Notebook/Cranial nerve development/2010/12/08

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Following a look at Malisha's sections we retrieved some H&E notes from Felipe. I staged an embryo at stage 21 and labeled it as RC003. Then I placed it in 30% sucrose and am waiting for it to sink. Meanwhile I set up a document called 'A brief look at neuroanatomy' and invited Malisha and Fabiana to be contributors for the document. I then put some work into the document to get it started. --Reuben Cutfield 18:02, 7 December 2010 (EST)

  • Emailed Satya asking about our Haematoxylin type - Is Gills 2 --Reuben Cutfield 18:27, 7 December 2010 (EST)
The Haematoxylin is purchased as a pure compound and then mixed using the Gills 2 protocol.

2:00pm I performed 4 separate H&E stains on the sections from embryo RC002 (A-E) and embryo MH002(F). I varied the length of time the slides were sitting in either the Haematoxylin or Eosin and recorded the times spent in each. The slides have been coverslipped and are ready for analysis.

(Note: Please provide details--MF Kubke 04:40, 20 January 2011 (EST))
H&E Staining of Embryo RC002 and MH002
H20 Hematoxylin Eosin Acid Alcohol Lithium Carbonate Alcohol
Slides A&B 10sec 30sec 1min 1sec 1sec 1sec
Slides C&D 20sec 45sec 1min 1sec 1sec 1sec
Slide E 5sec 45sec 1min15sec Ommitted 1sec 1sec
Slide F 5sec 25sec 1min15sec 1sec 2sec 1sec
(Note: The sections from MH002 fell of the slide and were not coverslipped. The slide was discarded. The remaining slides are in the folder labeled RC002.)
(Note: Can you please clarify your statement above? There is no mention of the sections falling on the embryo page, and it says there that I looked at the sections--MF Kubke 04:36, 3 February 2011 (EST))

Tomorrow Fabiana will do some cutting and staining to attempt to achieve some satisfactory results. --Reuben Cutfield 20:14, 7 December 2010 (EST)

(Note: I have located these slides and put them in a slide folder labeled 'Fabiana's Slides' which I will leave with my slide folders in the lab.--Reuben Cutfield 20:02, 20 January 2011 (EST))


Used an H& E stain to assess the history of the cryostat sections cut on Monday and Tuesday.

(Note: Please provide embryo details above--MF Kubke 04:42, 20 January 2011 (EST))

Feedback from fabiana : Large holes in the sections which are not expected with a stage 28 embryo (MH002) . At this point we cannot determine if it is an issue with the staining or the cut. More reading was done to " tweek" the staining procedure specifically for embryonic tissue--Malisha Hettiarachchi 20:30, 12 December 2010 (EST).

  • At this point the effect of freezing and using the tissue was discussed. MH002 had been frozen and used therefore the tissue damage could be mainly because of this.