Kubke Lab:Research/CND/Records2010-2011Summer/RC001

Cranial Nerve Development

Experiment

Embryo details

Species: Gallus gallus domesticus
Embryo Name: RC001
Embryo stage: ST22 Confirmed by Fabiana. (see Notebook Entry)
Staging description: Staged as a group exercise by Fabiana, Malisha and Reuben using the Hamburger and Hamilton (1951) staging system.
Fixation:
Cryoprotection: Cryoprotected in 30% Sucrose in 0.1M PBS
Material label and storage: Labeled as RC-001 and stored in sucrose in a vial prior to being cut. The slides produced by RC001 are in a slide folder in the lab labeled RC001.

Experiment details

Objective: To use the embryo in a staging demonstration (see Notebook Entry), cryoprotection practice and cryostat sectioning practice.
Procedure: 23/11/2011 see Notebook entry

• Embryo taken from 'stationery draw' where it was stored in PFA.
• As a group we staged the embryo and Fabiana labeled it as ST22, RC001.
• It was put into a vial filled with 30% sucrose, 0.1M PBS.

24/11/2010: Histology Training with Satya see Notebook entry

• The embryo was placed flat on a plastic mould and OCT was then added.
• The embryo was oriented 90° to the side of the block so that coronal sections could be obtained.
• The protruding OCT was cut from the sides of the block.
• The block was mounted onto the chuck at 90° 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	24th November 2010, 10:00am
Knife Angle	5°
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	Permafrost Original Menzel-Glaser Cut Edges, Frosted Ends
Plane of section
Number of slides	8
Observations	It is best to cut slowly through the specimen. The platform needs to be kept clean to prevent sections sticking. The steadier your hand when mounting the less damage to the tissue. Support the microscope slide with your second hand to keep it steady. The sections were not serial. Lots of sections were lost during experimentation as a result of experimenting with the cutting conditions. Quality of the sections were poor, as expressed by Fabiana.

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

Comments: Tissue from this embryo will not be used for histological analysis. RC001 was used for practice purposes only, it was not stained or coverslipped. 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 RC001.