Hi, I am looking for a protocol to preserve the structure of the skin during the criosectioning. I am trying to visualize the section of a skin sample, with particular interest in the hair follicles. I tried to freezese sample using liquid nitrogen after fixation with formaline, but I don't manage to visualize the follicles after this procedure.
The paraffine infiltration is not good in my case because I can not use the temperature required.
If you need any further details, please let me know!
Thanks!
Why do you use crosectioning? Criosectioning is used to avoid formaline fixation and paraffin embeding (FFPE). If you already have FFPE, your histology will be better than with criosectioning.
I agree with Ignacio, cryofreezing is a rapid solidifying method used when histological analysis is required quickly (e.g. if a biopsy is taken when a patient is in surgery) there is no need to fix the samples when you do this. Formalin draws all the water from the cells so you will not be able to visualise structures in the skin without paraffin infilration .
It is permissible to use formalin fixation for cryosectioning, but if you can help it, section your tissue right after you obtain it. If you are interested in performing immunohistochemistry on your tissue, then be mindful how long your it remains in contact with formalin or avoid it all together. Formalin fixation can affect your staining.
A standard formalin solution is mostly water, so I have to disagree with Hayley. To draw all the water out of a tissue, you are required to run it through a series of increasingly hydrophobic solutions - alcohols then xylenes. This technique is used for paraffin embedding.
Try dry ice instead of liquid nitrogen. Apply distilled water, then dry ice to lower the temperature. You may need a special plate for this method.
Start with thinner sections, 5 microns is ideal. If having problems, increase thickness gradually, 1 micron at a time. Once you know your ideal thickness, keep it consistent, as this will matter during staining.
Keep the blade angle at 5 degrees to the tissue. Again, increase the angle gradually if having problems.
Of course, make sure your blades are as sharp as possible.
Hair could be tough to work with. When it comes to microtomy, it has a consistency of steel wire, so it may slice apart your sections. So go slow and steady when moving your blade through the tissue.
Thank you for the answers.
Maxim, it is interesting what you said about the hair. In my case, I have problems even to find the follicles, they just disappear I am trying to understand what is the reason.
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