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