Kubke Lab:Research/CND/Records2010-2011Summer/RC006

Cranial Nerve Development

Experiment

Embryo details

Species: Gallus gallus domesticus
Embryo Name: RC006
Embryo stage: 20

(Note: embryo stage was not confirmed, should be assumed to be an approximation --MF Kubke 06:40, 17 January 2011 (EST))

Staging description:

• Allantois has vesicularized and is roughly the same size as the midbrain (indicating stage 20-21)
• Contour of mid-trunk region is a straight line and not curved enough to be stage 21.

Note: RC-006 Is a rather large embryo for its stage. Tissue appeared well preserved and hence it was selected for cutting.
Fixation: PFA
Cryoprotection:
Material label and storage:

Experiment details

Date: 10th January 2011 see Notebook entry
Objective:Use the embryo to create a series of cross-sectional images of the hindbrain to identify structural features.
Procedure:Complete a serial sectioning using the cryostat followed by a Nissl stain.

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

Cryostat
Knife
Day Cut
Knife Angle	1.5°C
Chamber Temp	-19°C
Object Temp	-19°C
Glass Slides	Superfrost PLUS
Plane of section
Number of slides	13
Observations

(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
Solution	Time	Comments
Defatting and rehydration step
Water
75% alcohol
95% alcohol
100% alcohol 1
100% alcohol 2
Xylene 1
Xylene 2
Xylene 3
Xylene 2
Xylene 1
100% alcohol 2
100% alcohol 1
95% alcohol
75% alcohol
Water
Staining and differentiation step
Water
Cresyl Violet
50% alcohol
70% alcohol acetic acid
95% alcohol
100% alcohol 1
100% alcohol 2
Xylene 1
Xylene 2
Xylene 3
Coverslip

Comments: The slides were kept in Xylene solution for 3 hours prior to coverslipping. The coverslipped slides have been put in a folder labeled RC-006 in the lab.

Results

The sections fell off the slides. The sections could be seen to move during coverslipping and there was tissue floating in the xylene solution. I stained three slides of Malishas which were mounted onto polysine slides and they did not fall off or move which indicated it was likely the slides which gave the problem.

Summary

This experiment showed us that we cannot use 'Permafrost' or 'Superfrost PLUS' slides to mount our tissue. The slides need to be coated in either gelatin or polysine to keep the sections adhering to them. I also think that we should not cut the embryo with a razor blade as the compression of the embryo trunk caused by the razor blade is ruining our sections. I think we should place the whole embryo in our mould and simply discard the most caudal and rostral regions and only mount hindbrain and surrounding region.

(Note: there is no reason why cutting the head off at the level of the wingbud should have any effect in your sections of the hindbrain. Once you place the whole embryo in the OCT and the OCT freezes, you will not be able to easily tell where the hindbrain begins. You will also have other problems associated with the curvature of the embryo that may lead to even more trouble. --MF Kubke 06:37, 17 January 2011 (EST))