Kubke Lab:Research/CND/Records2010-2011Summer/MH008
| Cranial Nerve Development | Experiment |
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.
- The embryo MH008 was staged according to the and Hamilton (1951) staging system.
- 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 |
| Knife | MX35 Premier +, 34 degrees, 80mm Thermo Scientific |
| Day Cut | 26th Jan 2011 see Notebook entry. |
| Knife Angle | 1.5° |
| Chamber Temp | -19°C |
| Object Temp | -19°C |
| Glass Slides | Gelatin-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 slides | 10, 157 sections |
| Observations | There were no sections lost during mounting. Every single slice of the embryo was mounted onto the microscope slides.
|
(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 | ||
| Solution | Time | Comments |
| Water | 5 Dips | |
| Cresyl Violet | 7 Minutes | |
| 50% alcohol | 2 minutes | |
| 70% alcohol acetic acid | 1 minute | |
| 95% alcohol | 2 minutes | |
| 100% alcohol 1 | 2 minutes | |
| 100% alcohol 2 | 2 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.
