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

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Cranial Nerve Development Experiment

Contents

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
SolutionTimeComments
WaterOmitted
75% alcohol 3min
95% alcohol5min
100% alcohol 1 10min
100% alcohol 2Omitted
Xylene 1 30mins
Xylene 2 Omitted
Xylene 3Omitted
Xylene 2 Omitted
Xylene 1 Omitted
100% alcohol 2 10min
100% alcohol 1 Omitted
95% alcohol10min
75% alcohol5min
Water 1min
Staining and differentiation step
SolutionTimeComments
Water 1min
Cresyl Violet 15min
50% alcoholOmitted
70% alcohol acetic acid Omitted
95% alcohol 1min
100% alcohol 1 2min
100% alcohol 2Omitted
Xylene 1 20-40min
Xylene 2 Omitted
Xylene 3Omitted
CoverslipInd.

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

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