Kubke Lab:Research/CND/Records2010-2011Summer/RC009

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Cranial Nerve Development Experiment

Contents


(Note: Reuben, you need to add dates and if possible approximate time for your procedures - Cannot link with the journal entries unless you do)

Embryo details


Species: Gallus gallus domesticus
Embryo Name: RC009
Embryo stage: ST24
Staging description: Staged by Reuben according to the Hamburger and Hamilton (1951) staging system. Stage not confirmed. See Notebook entry.

  • Was stored in PFA, staged in 0.9% saline and then replenished with PFA. Sectioned from PFA.
  • Elbow and knee joints are distinct. This may indicate that the embryo is greater than stage 24.
  • No toe demarcation visible. This may indicate that the embryo is less than stage 26.
  • Toe groove on developing leg not visible. This indicates that the embryo has not reached stage 25.
(Note: Not sure I understand what is the difference between the last two bullet points. Can you please clarify)
(Note: The demarcation of the toes begins with a groove. I began by trying to identify whether there was demarcation to rule out ST26 and then looked more carefully to see if I could identify the groove forming to rule out ST25)

Therefore the embryo is stage 24.
Fixation: PFA
Cryoprotection:
Material label and storage: The embryo sections have been mounted, stained and coverslipped with DPX. The slides are numbered and stored in the slide folder labeled RC009.

Experiment details


Objective: Analyze Nissl-stained serial sections of the embryo.
Procedure:

  • Staged the embryo according to Hamburger and Hamilton staging (1951).
(Note: This is an approximation as it was not checked)


Cryostat Sectioning see Notebook entry

  • Under a dissection microscope the head was cleaved from the tail using the dissection scissors at the level of the wing bud.
  • The embryo was embedded in OCT and frozen at -19°C (in the cryostat chamber), loaded onto a chuck and incubated for an hour.
  • Serial sections were cut and mounted onto the gelatin-subbed slides whilst time spent cutting the section and time spent in chamber before mounting were recorded into my lab-book.
  • The sections were dried and then Nissl-stained for visualization under a microscope.


{{KubkeLabCryostat| |Cryostat = Leica – CM3050S |Knife = MX35 Premier +, 34 degrees, 80mm Thermo Scientific |Day Cut = 18th Jan 2011 |Knife Angle = 1.5 ° |Chamber Temp = -19°C |Object Temp = -19°C |Glass Slides = Gelatin-subbedOriginal Menzel-Glaser Cut Edges, Frosted Ends. Subbed according to the Cold gelatin subbing protocol which included the pre-wash step. |Plane of section = Coronal |Number of slides = 13 |Observations = 7/161 sections weren't mounted due to either curling of the OCT or sticking of the section to the platform. The rest of the sections are in series. Sections: 45,99,118,132 146, 151 & 153 weren't mounted due to complications during cutting.

(Note: The quality of the sections looks good at first inspection)

Cresyl Violet staining- EVEN Slides For more informattion see Kubke_Lab:Nissl_Stain_Protocol

Date
Defatting and rehydration step
SolutionTimeComments
Water0 This step should not be omitted--MF Kubke 01:55, 20 January 2011 (EST)
70% alcohol 4mins
95% alcohol 4mins
100% alcohol 1 4mins
100% alcohol 2 0 This step should not be omitted--MF Kubke 01:55, 20 January 2011 (EST)
Xylene 1 10mins
Xylene 2 0 This step should not be omitted--MF Kubke 01:55, 20 January 2011 (EST)
Xylene 30 This step should not be omitted--MF Kubke 01:55, 20 January 2011 (EST)
Xylene 2 0 This step should not be omitted--MF Kubke 01:55, 20 January 2011 (EST)
Xylene 1 0 This step should not be omitted--MF Kubke 01:55, 20 January 2011 (EST)
100% alcohol 20 This step should not be omitted--MF Kubke 01:55, 20 January 2011 (EST)
100% alcohol 1 1min
95% alcohol1min
70% alcohol1min
Water0 This step should not be omitted--MF Kubke 01:55, 20 January 2011 (EST)
Staining and differentiation step
SolutionTimeComments
Cresyl Violet 5mins
70% alcohol1min
70% alcohol acetic acid 0
95% alcohol 1min
100% alcohol 1 1min
100% alcohol 20 This step should not be omitted--MF Kubke 01:55, 20 January 2011 (EST)
Xylene 1 Variable See below for times spent in xylene before coverslipping for each slide.
Xylene 2 This step should not be omitted--MF Kubke 01:55, 20 January 2011 (EST)
Xylene 3 This step should not be omitted--MF Kubke 01:55, 20 January 2011 (EST)
Coverslip

Cresyl Violet staining-ODD Slides For more informattion see Kubke_Lab:Nissl_Stain_Protocol

Solution Time Comments
Date
Defatting and rehydration step
SolutionTimeComments
Water 0This step should not be omitted--MF Kubke 01:55, 20 January 2011 (EST)
75% alcohol 4mins
95% alcohol4mins
100% alcohol 1 4mins
100% alcohol 2 2minsNoticed particles floating in the solution, worried it was tissue.
Xylene 1 10mins
Xylene 2 0 This step should not be omitted--MF Kubke 01:55, 20 January 2011 (EST)
Xylene 30 This step should not be omitted--MF Kubke 01:55, 20 January 2011 (EST)
Xylene 2 0 This step should not be omitted--MF Kubke 01:55, 20 January 2011 (EST)
Xylene 1 0 This step should not be omitted--MF Kubke 01:55, 20 January 2011 (EST)
100% alcohol 21min
100% alcohol 1 0 Chose to not use the second alcohol due to the amount of floating debris. This is not acceptable. You should make sure your solutions are ready before you start. This step should not be omitted--MF Kubke 01:55, 20 January 2011 (EST)
95% alcohol 1min
70% alcohol 1min
Water0 Didn't use water as I was still afraid that tissue was peeling off. This step should not be omitted--MF Kubke 01:55, 20 January 2011 (EST)
Staining and differentiation step
SolutionTimeComments
Cresyl Violet 5mins
70% alcohol1min OCT had dissolved and tissues HADN'T fallen off as previosuly thought.
70% alcohol acetic acid 0
95% alcohol 1min
100% alcohol 1 0This step should not be omitted--MF Kubke 01:55, 20 January 2011 (EST)
100% alcohol 2 1min
Xylene 1 Variable 3-34mins
Xylene 2 0This step should not be omitted--MF Kubke 01:55, 20 January 2011 (EST)
Xylene 30 This step should not be omitted--MF Kubke 01:55, 20 January 2011 (EST)
Coverslip Coverslipped using small coverslips. Found them easier and neater to work with.
(Note: Please incorporate this table below into your tables above in the comments section: eg, sections were kept in xylene 3 between x and y minutes during coverslipping)
Time Slide Spent in Xylene Prior To Coverslipping
Slide Number Time (mins)
1 3
2 7
3 5
4 9
5 7
6 11
7 10
8 13
9 13
10 16
11 18
12 34
13 2


Comments:I did not allow myself enough time to complete the sectioning of the embryo. I stopped halfway through the hindbrain. The other half of the hindbrain and head were discarded.

Results

Half of the hindbrain and head are missing from the slides as they were discarded not cut and the rest of the block was discarded

Images

Summary

The objective was not achieved as I did not allow enough time to complete the cutting of the embryo.


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