Hi all!
I am currently working on a project where we have gone from using mouse embryos (E7.5-9.0) to pig embryos (E.17-20). We have notices of late a lot of issues with the technical aspect of our histology (microscopic tears or folds), additionally, we have taken some older pig hearts (E33) that didn't embed well. The tears and folds are what I am most concerned about as I am unable to see them grossly while sectioning and due to the precious nature of the tissue I don't have the liberty to toss any sections.
The E33s seemed to still contain water and therefore they did not really "stick" to the cryoblock. I should note on these that the E33s were embedded in gelatin and the others in OCT.
The question of lipid content came up and I really couldn't find any practical advice. What I am looking for is your experience and suggestions. We are doing mainly IHC on the samples and want to make sure we don't lose any antigenicity.
We have experimented with a couple different fixatives: Bouins, Zambonis, 4% PFA. We typically use 4% PFA in our lab.
Any suggestions or resources would be greatly appreciated!
Thank you all so much for your continued advice and support, you are an awesome community to be part of!
In general everything, that decreases stability of the cutting-system leads to shatter, tears and folds. Also the cutting-angle is important. Too small --> folds, compression, only every second section; Too big --> shatter, "hopping" knife over the Surface
cutting temperature: too cold --> "hopping" knife, horizontal tears; too warm --> folds, wrinkels, compressed section
section thickness: too thin --> folds, only every second section, holes
I would try to surround the gelatine-blocks with additional OCT on thy cryo-chuck to increase stability. Maybe mount it again on the chuck with OCT.
If you don't see the failures on the gross section, it can't go as wrong.
Check cutting temp, angle and thickness. And have a look at Dr. Peters' Website. http://www.pathologyinnovations.com/
Hi
For sample preparation I can give you some tips:
I have worked with different tissue tips and sizes. Usually use 4% PFA or Zambonis fixative. Fixation is 24/48h after witch follows rinsing in PBS few hours to 24h. After that follows cryoprotection with sucrose. I usually use uprising gradient of sucrose from 5-10-15-20% for an 30' and end up in 30% until specimen sink at the bottom. Before embedding specimen need to be well dried with filter paper!
I consider this step very important because sucrose bind water from specimen and this uprising gradient allows gradual permeation of sucrose in specimen. That water can cause poor embedding in OCT medium.
Another important step is freezing. When your sample is in OCT medium it need to be frozen in container with cooled 2-methyl butane placed in liquid nitrogen or on dry ice.
While sectioning the angle of specimen, knife sharpness and temperature of specimen and camber are key parameters you need to monitor. Temperature oscillations usually causes problems with thickness and folding.
Hope this guidelines help you!
Marica
Hi all,
Thank you so much for your help and advice. I should have mentioned that we do also cryo protect via gradients (5, 15, 30). I think the extra OCT may help with stability, our lab members tend to embed with as little surrounding media as possible. It's something they like, and I have just sort of adjusted to. For instance in a cryomold that is 10mm x 10mm x 5mm, they have me embed and cut from the 5mm wide side. Would you all suggest that we amend this procedure and use the larger plane?
Additionally, our sections range in thickness from 10 um to 14 um. I used to cut at 7-10 um (cryo) however, our researchers requested otherwise. I find that 14 seems a bit thick, and 10 with the very tiny embryos (mouse E7.0-9.0) is too thin to get clean sections. What is a good way I can assess the best thickness for the sections and sell that to our investigators?
Thanks again!
Tanya
I think mounting the block with the larger plane on the chuck is better. If it is necessary to cut the smaller plane you can achieve more stability with additional oct.
The optimal thickness depends on the researchers aim and the needed stain.
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