Kubke Lab:Research/CND/Records2010-2011Summer/RC004

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Cranial Nerve Development Experiment

Embryo details


Species: Gallus gallus domesticus
Embryo Name: RC004
Embryo stage: ST25
Staging description: Approximation, unconfirmed,
Fixation: PFA
Cryoprotection: None.
Material label and storage: Slides in slide folder labeled RC004.

Experiment details


Objective: To investigate the best cryostat sectioning protocol. Use the tissue to establish Cresyl Violet staining protocol.
Procedure:

  • Embryo staged under a dissection microscope in .9% saline solution. See Notebook entry.
  • Embryo loaded into a plastic mould filled with OCT and incubated in the cryostat chamber at -17°C.
  • Embryo loaded onto chuck, embedded deeper in OCT and incubated in the metallic chuck holder at -17°C for 30minutes.
  • Sectioned the embryo whilst modifying the settings of the cryostat to obtain the best sections.
  • The sectioned were mounted onto polylysine slides and dried overnight.


Comments: Sections from slide 5 were deemed by Fabiana to be sufficient for study. The settings used to cut slide 5's sections were:

(Note: what happened to the information about the rest of the slides?--MF Kubke 04:48, 3 February 2011 (EST))
(Note: The settings for each section I cut during the experiment are in my notebook. Do you want it here too?)
(Note: This is where the information belongs. I would argue it is not very useful having to go back and forth to get information --MF Kubke 22:48, 3 February 2011 (EST))

|Cryostat = Leica – CM3050S |Knife = MX35 Premier +, 34 degrees, 80mm Thermo Scientific |Day Cut = 17/12/2010, 12:30pm (see Notebook entry) |Knife Angle = 1.5° |Chamber Temp = -19°C |Object Temp = -19°C |Glass Slides = Polylysine subbed slides. |Plane of section = Coronal |Number of slides = 1 |Observations = Was cutting the sections very quickly. Histology looked good under a microscope. }}

(Note: the cresyl violet staining in this page does not match the entries on the journal--MF Kubke 04:53, 3 February 2011 (EST))

Cresyl Violet staining
For more informattion see Kubke_Lab:Nissl_Stain_Protocol. Slides 1-5 were all stained together in one staining rack.

Date
Defatting and rehydration step
Solution Time Comments
Water Omitted
75% alcohol 3min
95% alcohol 5min
100% alcohol 1 10min
100% alcohol 2 Omitted
Xylene 1 30mins
Xylene 2 Omitted
Xylene 3 Omitted
Xylene 2 Omitted
Xylene 1 Omitted
100% alcohol 2 10min
100% alcohol 1 Omitted
95% alcohol 10min
75% alcohol 5min
Water 1min
Staining and differentiation step
Solution Time Comments
Water 1min
Cresyl Violet 15min
50% alcohol Omitted
70% alcohol acetic acid Omitted
95% alcohol 1min
100% alcohol 1 2min
100% alcohol 2 Omitted
Xylene 1 20-40min
Xylene 2 Omitted
Xylene 3 Omitted
Coverslip Ind.

Results

Sections were stained too intensely. They require less heavy staining. The results of this study were used to perform cryostat sections on following embryos.


Images

Summary