Kubke Lab:Research/CND/Records/RC009

=Embryo details=

Species: Gallus gallus domesticus Embryo Name: RC009 Embryo stage: ST24 Staging description: Staged by Reuben according to the Hamburger and Hamilton (1951) staging system. Stage not confirmed. See Notebook entry. Therefore the embryo is stage 24. Fixation: PFA Cryoprotection: Material label and storage: The embryo sections have been mounted, stained and coverslipped with DPX. The slides are numbered and stored in the slide folder labeled RC009.
 * Was stored in PFA, staged in 0.9% saline and then replenished with PFA. Sectioned from PFA.
 * Elbow and knee joints are distinct. This may indicate that the embryo is greater than stage 24.
 * No toe demarcation visible. This may indicate that the embryo is less than stage 26.
 * Toe groove on developing leg not visible. This indicates that the embryo has not reached stage 25.

=Experiment details=

Objective: Analyze Nissl-stained serial sections of the embryo. Procedure: Cryostat Sectioning see Notebook entry
 * Staged the embryo according to Hamburger and Hamilton staging (1951).
 * Under a dissection microscope the head was cleaved from the tail using the dissection scissors at the level of the wing bud.
 * The embryo was embedded in OCT and frozen at -19°C (in the cryostat chamber), loaded onto a chuck and incubated for an hour.
 * Serial sections were cut and mounted onto the gelatin-subbed slides whilst time spent cutting the section and time spent in chamber before mounting were recorded into my lab-book.
 * The sections were dried and then Nissl-stained for visualization under a microscope.

{{KubkeLabCryostat|
 * Cryostat = Leica – CM3050S
 * Knife = MX35 Premier +, 34 degrees, 80mm Thermo Scientific
 * Day Cut = 18th Jan 2011
 * Knife Angle = 1.5 °
 * Chamber Temp = -19°C
 * Object Temp = -19°C
 * Glass Slides = Gelatin-subbedOriginal Menzel-Glaser Cut Edges, Frosted Ends. Subbed according to the Cold gelatin subbing protocol which included the pre-wash step.
 * Plane of section = Coronal
 * Number of slides = 13
 * Observations = 7/161 sections weren't mounted due to either curling of the OCT or sticking of the section to the platform. The rest of the sections are in series. Sections: 45,99,118,132 146, 151 & 153 weren't mounted due to complications during cutting.

{{Note|The quality of the sections looks good at first inspection}}

Cresyl Violet staining- EVEN Slides ''For more informattion see Kubke_Lab:Nissl_Stain_Protocol

Cresyl Violet staining-ODD Slides ''For more informattion see Kubke_Lab:Nissl_Stain_Protocol

{{Note|Please incorporate this table below into your tables above in the comments section: eg, sections were kept in xylene 3 between x and y minutes during coverslipping}} Comments:I did not allow myself enough time to complete the sectioning of the embryo. I stopped halfway through the hindbrain. The other half of the hindbrain and head were discarded.

=Results=

Half of the hindbrain and head are missing from the slides as they were discarded not cut and the rest of the block was discarded

=Images=

=Summary=

The objective was not achieved as I did not allow enough time to complete the cutting of the embryo.