I am cutting rat brain sections at 20um at -20C. The tissue is cutting nicely out of the O.C.T solution, but I can not get a decent section to adhere to the slide.
I have included an image.
Hi Cody,
There could be a few different things going on. Since the tissue is cutting well, you likely have a good fix and they have sat in sucrose for enough time.
From the looks of your picture it seems you should just add a little PBS to the slice so that it kind of floats in the liquid on top of the slide. That should give you enough room to use a brush and make sure the tissue is flat. Then, while gently holding the slice in place with the brush, angle the slide so that the liquid begins to run down the slide, causing the slice to 'sit' down on to the slide again. There should be enough residual liquid to allow you to make some minor adjustments to get your tissue perfectly flat on to the slide and position it where you want.
Justin--
Thank you! I will give this a try and hopefully, I can improve my sectioning.
If that doesn't help, in my experience it is also worth to exchange the glass insert of the cryotome from time to time. Leica and other companies sell them. If the borders of the glass are not straight or the "gap" for the sample between metal base/blade and glass is too big, tissues crumble faster.
Tissue rolling is due to temperature differential. We typically hold tissue at -16 and cabinet at -20. For soft tissus like brain you may need to cut at lower temps. Remember cutting = friction x speed = heat! To maintain structure you need to cut into a colder environment. If the tissue heats up too much it will deform/roll. Drop your cabinet temperature by a degree or two and try again. Also remember thicker sections retain their temperature more than thinner, a quick fix sometimes is to increase section thickness.
I agree with Justin. I straighten out my slices with dH2O (some people use EtOH-- it just depends on what you'll be imaging for). If you're still having difficulty getting it onto the slide with ease or without rippling, I would suggest using a paint brush to dab a small amount of dH20/EtOH/PBS onto the portion of the slide that you're going to be placing your slice on. That will give you a small liquid surface between the tissue and the glass to make sure that your tissue is flat. Although, with this, you can get bubble, so be careful.
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