Kubke Lab:Research/CND/Records2010-2011Summer/RC003

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Cranial Nerve Development Experiment

Contents

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

Time spent in each staining solution
Slide # Alcohol Haematoxylin Acid Alcohol Lithium Carbonate Eosin
1 10s 1min 15s 1s 1s 30s
31min1min1s15s1min
55min45s2s10s1min
72min1min1s10s1min
(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
SolutionTimeComments
WaterOmitted for all slides
75% alcoholSlides 2,4: 2min Slide 6: 4min
95% alcoholSlides 2,4: 2min Slide 6: 4min
100% alcohol 1 Slides 2,4: 1min Slide 6: 2min
100% alcohol 2Slides 2,4: 1min Slide 6: 2min
Xylene 1 All slides 10min
Xylene 2 Omitted for all slides
Xylene 3Omitted 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% alcoholSlides 2,4: 30s Slide 6: 1min
75% alcoholSlides 2,4: 30s Slide 6: 1min
Staining and differentiation step
SolutionTimeComments
Water Slides 2,4: 5s Slide 6: 10s
Cresyl Violet Slide 2: 2min Slide 4: 4min Slide 6:6min
50% alcoholOmitted for all slides
70% alcohol acetic acid Omitted for all slides
75% alcoholSlides 2,4: 2min Slide 6: 4min
95% alcoholSlides 2,4: 2min Slide 6: 4min
100% alcohol 1 Slides 2,4: 1min Slide 6: 2min
100% alcohol 2Slides 2,4: 1min Slide 6: 2min
Xylene 1 Until Coverslipped <30mins
Xylene 2 Omitted for all slides
Xylene 3Omitted 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))

Images

Summary

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