Hi everyone,
I am using a sliding microtome to slice some brains I have been collecting for the purpose of immunofluorescence (the brains have been PFA/PBS perfused, then post-fixed overnight in 4% PFA, and then transferred to 30% sucrose for a few days to be sliced at 60 microns). Some of my sections imaged under confocal microscopy appear to show the tracks of the microtome knife (I see this when I take a tile scan of the whole coronal section I am imaging), other sections do not show tracks of the microtome knife.
For reference, after the brains are cryoprotected and sink in sucrose, I place a drop of OCT on the microtome stage (that is pre-cooled with dry ice), and place the brain with the cerebellum cut on the stage (to make a flat surface on which the brain can be leveled with the stage to cut leveled sections...). I then use 30% sucrose, and with dry ice still on the stage, cover the brain with a sucrose column using a micropipette (a tip I received from research gate) to make some sort of mold to help with cutting the brain. This helps produce free-floating sections, which I use to perform free-floating immunofluorescence staining.
I am wondering if anyone has any suggestions and advice for better slicing with a sliding microtome to avoid knife tracks in my sections. Reading on this a bit, it seemed that the clearance angle of the knife needed to be adjusted. But even after trying to adjust it, I still find that some sections have knife tracks in them.
The microtome I am using is similar in the make/model to this one : https://www.fishersci.ca/shop/products/hm-430-sliding-microtome-3/22050855
Many thanks,
Rania
HI Rania,
This usually happens when the tissue is too cold. If the tissue is too cold, it is harder and results in the knife skipping across the tissue instead of slicing cleanly through it, resulting in the tracks you see under the microscope. You can test this in theory while slicing to see if it is your issue. Slice until you start to see this issue develop. When you see it, simply put your gloved finger on the tissue for a few seconds, in essence warming up, and then take another slice and look at it under the microscope. If the issue disappears, then it is the temperature that is affecting the quality of your samples. As a tip, I always look at every 10th slice or so under the microscope or at least up to the light to make sure everything is ok with the sample before slicing too much farther into the tissue. This will ensure you don't waste valuable samples with a poor cutting procedure. If you could attach a photo of what you are seeing when you look under the microscope, it will help us to make sure we answer your question accurately.
Best,
Renee
Hi Renee,
Thank you so much for your insightful help as always. I'm going to try your suggestion and hope it is just the tissue being too cold when it's being cut. The knife definitely doesn't always feel smooth when I am sliding it across the brain, but I did not think it would be the temperature of the brain- I actually thought it could be the angel of the knife with the stage or that my tissue needs to be on a more planar surface..
Here are some pictures from when I was practicing tile scans on a Zeiss confocal microscope (the staining on the slices is not that great, but I got feedback from the microscopy tech that the tissue has tears and knife tracks..
Thank you for your helpful insights and advice.
Rania
Hi Rania,
The second and third picture definitely show evidence of what we call "chatter" caused by the tissue being too cold (the lines representing knife marks). However, I don't think the holes or black spots you are seeing in the first picture are from the temperature. These look more like freezing artifact or fixation issues. What are you making your sucrose solution in? PBS? ddH2O? On another note, the angle of the blade can definitely impact your slicing. However, in my opinion, incorrect knife angle causes uneven cutting or damage, not the issues I see here. Here is a good review of knife angle and the issues it can cause:
https://www.leicabiosystems.com/knowledge-pathway/knife-angle-in-microtomy/
Definitely think you will get less of these artifacts with a warmer specimen.
Renee
Hi Renee,
Thanks so much for your feedback. I make my 30% sucorse solution in 1X PBS not ddH2O. I have tried your tip and warmed up the tissue with my finger and have noticed the knife slide smoother across the brain when cutting. I plan to stain my sections soon, so hoping that they return images with less of these artifacts.
Thanks,
Rania