Talk:Kubke Lab:Protocols/Cryomicrotomy

Page created from notes that were taken by Reuben Cutfield --Reuben Cutfield 20:19, 9 December 2010 (EST) whilst using the cryostat in the Histology section of Anatomy with Radiology department, University of Auckland, managed by Satya Amirapu. Under the guidance of Satya Amirapu (Senior histologist, University of Auckland) and Fabiana Kubke (supervisor) the cryomicrotomy technique was learnt and applied.

Suggestions

Using a razor blade to mount your sections:

A cool razor blade with a handle made of tape may be used to keep the sections flat following cutting. Place the razor blade on the platform so that the incoming sections are fed under the blade. This technique has not been confirmed to damage the tissue but sections should be carefully analysed when using this technique. This allowed us to cut sections up to 25um thick whilst mounting remained easy.

Not sure this is necessary. If the guideplate and knife angle are adjusted and enough time is given for the sections to sit before lifting the guideplate there should not be significant curling. I am afraid that this has two problems: One that the temperature of the cryostat will be less stable as the door will need to be open at most times, and there is danger of damaging the sections. --MF Kubke 22:35, 9 December 2010 (EST)

Factors affecting the quality of sections:

1. The thinner a section is the less likely it is to curl
2. The OCT will be too warm to cut properly upwards of an OT (object temperature) of -16 degrees
3. Lowering the temperature appeared to make only subtle differences in which the sections are not pressed under the glass as flat making mounting less neat
4. Warmer cutting temperatures may create less crystals than at colder temperatures
5. If you need to stop cutting keep the tissue in the freezer compartment rather than taking it out, thawing it or putting it into a deep freeze. This may prevent the formation of crystals
6. There is no ideal temperature to section at and experimentation is required. There were no obvious differences when cutting between 17-30 degrees although each tissue will be different

Other issues

The tissues sectioned were chick embryos fixed in paraformaldehyde between stages 20-30 (Hamburger and Hamilton Staging). It should be noted that New Zealand is very humid and these notes were produced during the summer period. Hence the temperatures and observations may not be relevant to cooler and dryer regions. The sections were cut manually using the rotator. This method was selected as opposed to the motorized version due to the delicate nature of the tissue and the thickness of the slices required (10-25um).