Kubke Lab:Research/CND/Records2010-2011Summer/MH008

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Cranial Nerve Development Experiment

Contents

Embryo details


Species: Gallus gallus domesticus
Embryo Name: MH008
Embryo stage: ST19 Confirmed by Fabiana (supervisor). (see journal entry)
Staging description: Staged by Fabiana.
Fixation:
Cryoprotection:
Material label and storage:

Experiment details


Objective:To complete a serial coronal sectioning of the embryo rostral to the wingbud prior to staining with Cresyl Violet and coverslipping for histological and cytological analysis of the stained sections.
Procedure: 26th Jan 2011 see Notebook entry.

  • 11:30am Using dissection scissors and under a dissection microscope I cut the embryo just rostral to the wing bud
  • The head region of the embryo was then gently replaced into a vial filled with PFA using forceps.
  • A small plastic mould was half filled with OCT and then, using a blade, the embryo was very carefully laid on top of the OCT from a Petri dish containing PFA.
  • Using forceps the embryo was oriented such that the hindbrain ran parallel to the sides of the mould so that coronal sections could be made
  • Bubbles in the OCT were removed with the forceps.
  • 12:05pm Incubated the block at -19°C in the cryostat chamber.
  • 12:30pm The block was oriented 90° to a chuck and stuck on by freezing OCT between the two contacting surfaces.
  • OCT was slowly built up either side of the block inside the cryostat chamber.
  • The chuck was inserted into the metallic chuck holder and incubated at -19°C for 30 minutes.
  • 1:30pm Began trimming the block
  • Serial sections of the specimen were cut and mounted onto microscope slides and dried overnight in a staining rack, in a fume hood, wrapped in tin foil. See the Cryomicrotomy page for information on how this was done.
  • The sections were then stained using Cresyl Violet (see below for the time the slides spent in each solution). See Notebook entry. Only the staining and differentiation step was used.
(Note: I was told that the defatting step was unnecessary and omitting it saves time. I will assess the quality of staining to determine if this is true.)


Cryostat Sectioning Cryostat settings (for a more detailed protocol visit Kubke_Lab:Cryomicrotomy)

Cryostat Leica – CM3050S
KnifeMX35 Premier +, 34 degrees, 80mm Thermo Scientific
Day Cut26th Jan 2011 see Notebook entry.
Knife Angle1.5°
Chamber Temp-19°C
Object Temp-19°C
Glass SlidesGelatin-subbed Original Menzel-Glaser microscope slides with cut edges and frosted Ends. Slides were subbed using the Cold gelatin subbing protocol including the pre-wash procedure, see Notebook entry 24/1/11.
Plane of section
Number of slides10, 157 sections
ObservationsThere were no sections lost during mounting. Every single slice of the embryo was mounted onto the microscope slides.
  • Section 6 showed pinching.
  • Section 26 might have overlapped with some OCT from a neighbouring section on the right side of the section.
  • Section 63 may have had the knife touch it slightly.
  • Section 78 might have had OCT overlap too.
  • Section 100 I didn't wait long enough before mounting.

(Include in your observations, eg ,were the sections serial, was any section lost, was quality assessed, etc)



Cresyl Violet staining
For more informattion see Kubke_Lab:Nissl_Stain_Protocol

Date
Defatting and rehydration step
Step Ommitted
Staining and differentiation step
SolutionTimeComments
Water 5 Dips
Cresyl Violet 7 Minutes
50% alcohol2 minutes
70% alcohol acetic acid 1 minute
95% alcohol 2 minutes
100% alcohol 1 2 minutes
100% alcohol 22 minutes
Xylene 1 2minutes
Xylene 2 2minutes
Slide Number Time spent in Xylene 3 (mins)Comments
1 11
2 19
3 7
4 17
5 15
6 14
7 13
8 10
9 8
10 5



Comments: Staining is lighter than usual. Appears to show more contrast.

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