Template:KubkeLabEmbryo

Cranial Nerve Development

Experiment

Embryo details

Species: Gallus gallus domesticus
Embryo Name: RC-006
Embryo stage: 20
Staging description:

• Allantois has vesicularized and is roughly the same size as the midbrain (indicating stage 20-21)
• Contour of mid-trunk region is a straight line and not curved enough to be stage 21.

Note: RC-006 Is a rather large embryo for its stage. Tissue appeared well preserved and hence it was selected for cutting.

Experiment details

Objective:Use the embryo to create a series of cross-sectional images of the hindbrain to identify structural features.
Procedure:Complete a serial sectioning using the cryostat followed by a Nissl stain.
Comments: Cryostat settings: 1.5 degrees knife angle, -19 degrees C chamber and object temperature of the cryostat. The slides were kept in Xylene solution for 3 hours prior to coverslipping. The slides used to mount the sections were 'Superfrost PLUS' slides.

Results

The sections fell off the slides. The sections could be seen to move during coverslipping and there was tissue floating in the xylene solution. I stained three slides of Malishas which were mounted onto polysine slides and they did not fall off or move which indicated it was likely the slides which gave the problem.

The coverslipped slides have been put in a folder labeled RC-006 in the lab.

Summary

This experiment showed us that we cannot use 'Permafrost' or 'Superfrost PLUS' slides to mount our tissue. The slides need to be coated in either gelatin or polysine to keep the sections adhering to them. I also think that we should not cut the embryo with a razor blade as the compression of the embryo trunk caused by the razor blade is ruining our sections. I think we should place the whole embryo in our mould and simply discard the most caudal and rostral regions and only mount hindbrain and surrounding region.