Kubke Lab:Research/CND/Records2010-2011Summer/RC003: Difference between revisions
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=Results= | =Results= | ||
Cresyl Violet staining gave better results than H&E staining. Based on the results we will discontinue staining with H&E. | Cresyl Violet staining gave better results than H&E staining. Based on the results we will discontinue staining with H&E. | ||
{{Note|You seem to have used different timing for different slides for CV, were there any differences in staining?--[[User:M Fabiana Kubke|MF Kubke]] 04:43, 3 February 2011 (EST)}} | |||
=Images= | =Images= | ||
Revision as of 02:43, 3 February 2011
| Cranial Nerve Development | Experiment |
Embryo details
Species: Gallus gallus domesticus
Embryo Name: RC003
Embryo stage:ST21
Staging description:Confirmed by Malisha and Fabiana.
Fixation: PFA
Cryoprotection:30% Sucrose solution.
Material label and storage:Slides kept in slide folder labeled RC003.
Experiment details
Objective: Use the embryo to practice sectioning in the cryostat. Investigate the settings which will provide the best quality sections for studying the histology of the tissue. Use the tissue to evaluate the best staining protocol to use to visualize the tissue histology.
Procedure:
- Embryo staged under a dissection microscope following removal of PFA, replenished with .9% saline solution. See Notebook entry.
- Embryo was placed in 30% sucrose solution and allowed to sink.
- Fabiana cut the embryo in the cryostat and mounted the sections onto polylysine slides. See Notebook entry.
- I performed an H&E stain on the odd numbered slides and a Cresyl Violet stain on the even numbered slides. See Notebook entry.
Comments:
H&E staining
For more information see Kubke Lab:Protocols/Hematoxylin Eosin
| Slide # | Alcohol | Haematoxylin | Acid Alcohol | Lithium Carbonate | Eosin |
|---|---|---|---|---|---|
| 1 | 10s | 1min 15s | 1s | 1s | 30s |
| 3 | 1min | 1min | 1s | 15s | 1min |
| 5 | 5min | 45s | 2s | 10s | 1min |
| 7 | 2min | 1min | 1s | 10s | 1min |
- (Note: can you please include the entire procedure for H+E, please. I assume that the sections were dehydrated prior to coverslipping?--MF Kubke 04:42, 3 February 2011 (EST) )
Cresyl Violet staining
For more informattion see Kubke_Lab:Nissl_Stain_Protocol
| Date | ||
| Defatting and rehydration step | ||
| Solution | Time | Comments |
| Water | Omitted for all slides | |
| 75% alcohol | Slides 2,4: 2min Slide 6: 4min | |
| 95% alcohol | Slides 2,4: 2min Slide 6: 4min | |
| 100% alcohol 1 | Slides 2,4: 1min Slide 6: 2min | |
| 100% alcohol 2 | Slides 2,4: 1min Slide 6: 2min | |
| Xylene 1 | All slides 10min | |
| Xylene 2 | Omitted for all slides | |
| Xylene 3 | Omitted for all slides | |
| Xylene 2 | Omitted for all slides | |
| Xylene 1 | Omitted for all slides | |
| 100% alcohol 2 | Slides 2,4: 15s Slide 6: 30s | |
| 100% alcohol 1 | Slides 2,4: 15s Slide 6: 30s | |
| 95% alcohol | Slides 2,4: 30s Slide 6: 1min | |
| 75% alcohol | Slides 2,4: 30s Slide 6: 1min | |
| Staining and differentiation step | ||
| Solution | Time | Comments |
| Water | Slides 2,4: 5s Slide 6: 10s | |
| Cresyl Violet | Slide 2: 2min Slide 4: 4min Slide 6:6min | |
| 50% alcohol | Omitted for all slides | |
| 70% alcohol acetic acid | Omitted for all slides | |
| 75% alcohol | Slides 2,4: 2min Slide 6: 4min | |
| 95% alcohol | Slides 2,4: 2min Slide 6: 4min | |
| 100% alcohol 1 | Slides 2,4: 1min Slide 6: 2min | |
| 100% alcohol 2 | Slides 2,4: 1min Slide 6: 2min | |
| Xylene 1 | Until Coverslipped <30mins | |
| Xylene 2 | Omitted for all slides | |
| Xylene 3 | Omitted for all slides | |
| Coverslip | Ind. | |
Results
Cresyl Violet staining gave better results than H&E staining. Based on the results we will discontinue staining with H&E.
- (Note: You seem to have used different timing for different slides for CV, were there any differences in staining?--MF Kubke 04:43, 3 February 2011 (EST))
