Kubke Lab:Research/CND/Records2010-2011Summer/RC002

Cranial Nerve Development

Experiment

Embryo details

Species: Gallus gallus domesticus
Embryo Name: RC002
Embryo stage: ST23 Not Confirmed by Fabiana.
Staging description: Staged by Reuben according to the Hamburger and Hamilton (1951) staging system.

• Eye shows faint pigmentation around its border (indicating it has surpassed ST20)
• The wing and leg buds growth progress matches ST23.

Fixation:
Cryoprotection: Not cryoprotected. PFA was replenished following staging the embryo in 0.9% saline solution.

(Note: This is not cryoprotection --MF Kubke 02:56, 27 January 2011 (EST))

Material label and storage: Labeled as RC-002 and stored in PFA in a vial prior to being cut. The slides produced by RC002 are in a slide folder in the lab labeled RC002. The coverslip mountant was DPX.

(Note: the DPX information is part of your procedures, please add to where you describe the coverslipping procedure --MF Kubke 02:56, 27 January 2011 (EST))

Experiment details

Objective: To section the embryo in the cryostat, experiment with the cutting technique and protocol and to assess whether omitting the cryoprotection step delivers better results.
Procedure:
2/12/2010 Staging see Notebook entry

• Embryo taken from 'stationery draw' where it was stored in PFA.
• The embryo was tipped into a dissection dish and the PFA was sucked out with a vacuum.
• The embryo was bathed in 0.9% saline.
• Following staging the embryo was returned, using forceps, to the vial and replenished with fresh PFA.

03/12/2010: Cryostat Sectioning see Notebook entry

• The embryo was placed flat on the lab bench and using a razor blade the embryo was cut.

(Note: The embryo was improperly cut too rostrally and the hindbrain was severed in half.)

• The embryo was placed flat on a plastic mould and OCT was then added.A mark was made on the mould to show the orientation of the embedded embryo.
• The embryo was oriented 90° to the side of the block so that coronal sections could be obtained.
• A chuck was placed in the cryostat chamber at -24°C and allowed a few minutes to cool down before a small amount of OCT was added and the hardening of the OCT was used to adhere the block to the chuck.
• A mark was made on the block corresponding to the line drawn on the metallic chuck holder, so that the block could be re-aligned if it had to be moved from the metallic chuck holder.
• The embryo was then sectioned in the cryostat.

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	December 3rd 2010, 1:30pm see Notebook entry.
Knife Angle	Varied throughout experiment.
Chamber Temp	The temperature was changed throughout the experiment as part of the experiment. See each individual slide for information on the temperatures used.
Object Temp	The temperature was changed throughout the experiment as part of the experiment. See each individual slide for information on the temperatures used.
Glass Slides	Polylysine-subbed slides by Menzel-Glaser.
Plane of section
Number of slides	5
Observations	The OCT may be sculpted into different shapes which provide variable likelihoods of curling during cutting. A candy wrapper shape demonstrate no effect on curling. Using excessive OCT also didn't prevent curling. You may use a razor blade placed underneath the glass to prevent curling. (Note: This may damage the tissue and is not recommended) The sections were not serial. Lots of sections were lost during experimentation as a result of experimenting with the cutting conditions.

(Include in your observations, eg ,were the sections serial, was any section lost, was quality assessed, etc)

06/12/2010: H&E Stainingsee Notebook entry.

(Note: Please add this information in this record page --MF Kubke 02:56, 27 January 2011 (EST))

Comments: A Hematoxylin and Eosin stain was performed on the embryo. The sections did not fall off the slides. They were coverslipped following staining. According to Satya (Senior Histologist) the sections needed to spend more time in eosin. The staining was heaviest with the thicker sections. Tissue from this embryo will not be used for histological analysis. Quality of the mounted sections were poor, as expressed by Fabiana.

Results

It appears that sectioning the embryo at 20microns gave the best results in this experiment. Using the blade to aid in mounting produced conflicting results and it was not clear whether the technique gave better results. It was thought that it was likely the blade was damaging the tissue. The polylysine subbing allowed the sections to adhere to the glass.

Summary

RC002 was used for experimentation purposes only. The settings of the cryostat and the cutting techniques were altered to learn the effects the settings had on the quality and efficiency of the sections produced. No further work will be done with RC002.