Kubke Lab:Research/CND/Records/RC002

=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. Fixation: PFA was replenished following staging the embryo in 0.9% saline solution. Cryoprotection: Not cryoprotected. 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.
 * Eye shows faint pigmentation around its border (indicating it has surpassed ST20)
 * The wing and leg buds growth progress matches ST23.

=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: Staging see Notebook entry 2/12/2010 Cryostat Sectioning see Notebook entry 03/12/2010
 * 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.
 * The embryo was placed flat on the lab bench and using a razor blade the embryo was cut.
 * 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.
 * Sections were stained with Cresyl Violet solution and then coverslipped using DPX mountant.

H&E Staining Different sections from RC002 were stained with H&E stain on the 6th (see Notebook entry 06/12/2010.) and 8th (see Notebook entry 08/12/2010.) of December 2010.

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.

=Images= =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.