Kubke Lab:Research/CND/Records/RC015

=Embryo details=

Species: Gallus gallus domesticus Embryo Name: RC015 Embryo stage: ST20 + Confirmed by Fabiana (supervisor). Staging description: Staged on the 20th Jan 2011 10:00am, see Notebook Entry Fixation: PFA Cryoprotection: None. Material label and storage: The sectioned, mounted, stained and coverslipped (in DPX) embryo is labeled RC015 and stored in a slide folder in the lab.
 * Tail bud points forwards
 * Leg and wing buds roughly the same size (indicative of an embryo ST20-ST21)
 * Faint pigmentation present in the eye (pigmentation begins to develop at ST20)
 * Maxillary visceral arch is distinctly larger than the Mandibullary which extends to midline of eye (indicating ST20-ST21)
 * 2nd Visceral arch distinct. (Indicating ST20-ST21).

=Experiment details=

Objective:To complete a serial coronal sectioning of the embryo rostral to the wingbud prior to staining with Cresyl Violet and coverslipping for histological and cytological analysis of the stained sections. Procedure: 20th Jan 2011, 10:15am (see Notebook entry)
 * The embryo RC015 was staged according to the Hamburger and Hamilton (1951) staging system.


 * 11:30am Using dissection scissors and under a dissection microscope I cut the embryo immediately rostral to the wing bud


 * The cryostat was set to OT: -19°C, CT: -19°C and allowed to equilibrate to the new temperature (it was previously set to OT:-17°C, CT: -17°C.
 * The head region of the embryo was then gently replaced into a vial filled with PFA using forceps.
 * A small plastic mould was half filled with OCT and then, using a blade, the embryo was very carefully laid on top of the OCT from a Petri dish containing PFA.
 * Using forceps the embryo was oriented such that the hindbrain ran parallel to the sides of the mould so that coronal sections could be made

21st Jan 2011, 9:30am see Notebook entry
 * Bubbles in the OCT were removed with the forceps.
 * 11:55am Incubated the block at -19°C in the cryostat chamber.
 * 12:15pm The block was oriented 90° to a chuck and stuck on by freezing OCT between the two contacting surfaces.
 * OCT was built up either side of the block inside the cryostat chamber.
 * The chuck was inserted into the metallic chuck holder and incubated at -19°C for one hour.
 * 1:15pm Began trimming the block but decided once I had trimmed to the embryo that it was not centered properly and so more OCT was built up on the side lacking OCT to centre the tissue in the OCT mould.
 * The assembly was incubated in the metallic chuck holder for 30 minutes prior to cutting to allow it to harden.
 * Serial sections of the specimen were cut and mounted onto microscope slides and dried overnight in a fume hood.
 * Slides 1,3,5,7,9,10,11 & 15 were cut by Reuben and Slides 2,4,6,8,12,13,14 were cut by Malisha.
 * A Cresyl Violet stain was then done by Reuben on the tissue sections prior to coverslipping and storing the slides in the folder labeled RC015.

Cresyl Violet staining For more informattion see Kubke_Lab:Nissl_Stain_Protocol

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=Results=

=Images=

=Summary=