Presence of Ice Artifacts

[KCalvin]

Hi all:

Just wanted some general suggestions and feedback on how can I eliminate what appears to be holes in my sectioned brain tissue due to ice artifacts.

Brain was perfused in 4% PFA and fixed in 4% PFA, cryoprotected in 20%/ 30% sucrose and then placed in mold with OCT before placing in the -80 freezer. The tissue was then placed in the -18 cryostat for a 20 min "equilibration".

When cut, the sections appears to have many small holes, in which I believe are ice artifacts. How can I work to eliminate these artifacts? And, off hand thought, will having significant ice artifacts on the tissue affect/contribute/cause the tissues to slide off the slides when I put them through a cresyl violet stain protocol?

Thanks for all the comments and help!

[CanuckPhD]

The issue is that you froze your tissue in the -80, which in my experience always leads to ice crystal formation due to the slow freezing. If you search on this site I have posted a detailed protocol on how to freeze rodent brains using isopentane and a dry ice bath.

If there are is lots of ice crystals in the tissue the morphology will preclude any analysis of neuronal number, health or IHC.

[KCalvin]

Thank you for the input, really appreciate it.

And while we are on the topic, I was wondering if there may be other potential areas of error that may result in such characteristic tears/breaks in the tissues. Quite the novice myself, I'm not terribly confident that what I am seeing are in fact ice artifacts or rather something else. Perhaps errant cryostat settings/procedure. I bring this up because I have had minimal, if any, breaks/tears in my tissues in the recent past when I first started slicing brains, but it inexplicably began appearing now. The point being is that I havent's changed my slow freezing protocol but I have been playing around with cryostat settings.

Thanks once again.

[CanuckPhD]

The cutting temperature for brain is also important. It cuts best at about -15 Celsius (similar to spleen). I have found that if it is too cold there is "chattering" artefact.

[KCalvin]

I recently tried a fast freezing protocol, and due to the ready availability of dry ice and 100% ethanol, I used the combo as opposed to isopentane. Two questions regarding this: 1. Is there an appropriate ratio of each that we must adhere to and 2. Is there an ideal time frame in which the freezing should take place. I think you mentioned elsewhere that the temperature in the isopentane case needs to be around-55 and -60 since too cold results in brittleness and too warm results in holes. Are there similar restrictions for the dry ice and ethanol that you are aware of.

[CanuckPhD]

What we usually do is make a slurry of about 50:50 dry ice and absolute ethanol in a large styrofoam box and then place a 500 ml plastic beaker with about 200 mls of isopentane in the center. We let it get to -55 and then put in a test block of OCT (a mold with no tissue just OCT). If this freezes in 1 minute then the proper temperature has been reached. We then time all of the freezing and take the tissues out after 1 minute, making sure that the temperature does not get too cold or too warm.

I must admit the test block is a hold over from when I didn't have a thermometer that went to -80, so did this instead.