Kubke Lab:/Notebook/Cranial nerve development/2011/01/14

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General Entries

  • I Have created a template to login the information of the embryos used for experiments.

Please use the following template:


Please create a single page for each embryo and name the pages as:


  • I have also created a template to share what each of us is reading. Please use the following Template:

I think the best thing is to describe the specifics of the paper on the front page and use the talk page to actually discuss the paper. Please name this pages with the following format:


(add a, b, c, etc at the end as necessary)

Personal Entries


  • 9:00 AM: Began preparation for cryostat session by staging embryo MH005
  • 12.50 PM: Began cutting further details can be found here. --Malisha Hettiarachchi 04:57, 16 January 2011 (EST)
(Note: Please, fill in the details in the embryo record sheet --MF Kubke 05:43, 20 January 2011 (EST))

Plan for monday: Stain 11 slides from MH005 and Fabiana's slides.--Malisha Hettiarachchi 04:57, 16 January 2011 (EST)


  • 10:00am Nissl Staining of embryo RC006 (ST20) (cut yesterday).--Reuben Cutfield 17:20, 13 January 2011 (EST)
    • Also staining three slides for Malisha.
(Note: Please provide embryo number and fill in the details in the embryo sheet --MF Kubke 05:46, 20 January 2011 (EST))
  • 11:30am Finished staining. Slides ready for coverslipping.
  • 12:15pm Disposed of the 75%, 95% and 100%(2) EtOH solutions. The solutions were replenished with EtOH from a sealed bottle:-Reuben Cutfield 18:42, 13 January 2011 (EST)

Concentrations used to make solutions for Nissl staining. (Total V=800mls)
Concentration EtOH(mls) dH2O(mls)
70% 560 240
95% 760 40
100% 800 0

Results: The sections were not adhering to the slides as well as they were during the staining of previous embryos. We had run out of the 'Polylysine' subbed slides and this time used the 'Superfrost Plus' slides, which Satya said would be suitable. The sections were moving during coverslipping. I eventually managed to coverslip the final slides gently enough to keep them from moving but under a microscope it is evident that the integrity of the micro-structure was being lost. Small fragments of the sections were also present in the xylene solution prior to coverslipping indicating that the problem was present prior to coverslipping.

(Note: Reuben, you guys had already had this problem with the sections not sticking on permafrost slides, which is why you changed to polylysine. (I am also not sure that leaving the sections sitting in xylene would have helped). Can you please log in the amount of time that your sections sat in xylene prior to coverslipping (please enter all the details in the RC006 page)--MF Kubke 22:34, 13 January 2011 (EST))

Staining was however good and for the next embryo will use the same protocol:

  • 4 minutes in the alcohols each
  • 20 minutes in Xylene
  • 5 minutes in cresyl violet
  • 10-20 seconds in each alcohol during second dehydration (keeping an eye on the intensity).

Lab Notes:

  • Xylene was filtered and topped up.
  • 100%EtOH(1) was replenished with new 100% since it was murky and had tissue floating in it.
  • 75%EtOH and 95%EtOH were clearly diluted and a small amount of 100% EtOH was added to raise the concentration back within the range we require for each.
Does this mean that the concentration of alcohol is currently unknown? If so, please dispose of that alcohol and fill the containers with the appropriate alcohol.
This has now been done.
The staining solutions are now all new or have been filtered except for the cresyl violet solution. --Reuben Cutfield 18:42, 13 January 2011 (EST)
  • Tap Water was replenished.
  • The silica beads turned orange and have been returned to our lab above the dissection microscope. --Reuben Cutfield 19:42, 13 January 2011 (EST)


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