I have attached some photos of what I am observing when I cryosection P0 mouse brains. I have had this issue for a while, and have made many adjustments to fix it, but no luck....
For reference, here is the protocol I have been using:
-Dissect out whole P0 mouse brain at RT in 1x pbs
-drop fix brain in 4% pfa on rocker for one day in 4d fridge (I have used several different pfa solutions--lead to no resolution)
-graded sucrose solutions (15-25-30% sucrose in 1x pbs) over the span of two days in 4d on rocker (at the end of the 2 days, brains have completely sunk)
-embed brains in OCT using a mold in dry-ice bucket with lid (ensuring there is no interface between the brain and oct, i.e. drying off sucrose with KimWipe before swirling brain in oct)
-storing brain in -80 until cryosectioning
-place brain in cryostat ranging from -18 to -20 (i've tried different cutting temps--no luck)
for 30 mins prior to sectioning
After all of this, my sections themselves are coming out flat, but the area containing the tissue is either bunched up, or has a large hole. Also, the brain on the mounting chuck looks dry/flaky. I originally thought that sucrose was not diffusing through the tissue and displacing the pfa, so I have adapted a graded sucrose protocol, which I've done at RT and 4d and no luck. I have tried thawing with thumb before each section, and no luck...
Really any help is appreciated!!
Any chance that a 1xpbs solution with a ph of 7.7 would be an issue? I figured no because it's a buffer, but possible this could be the issue ?
The most obvious answer to the holes part of your question is that your tissue isn't completely infiltrated with the embedding medium, similar to what occurs in paraffin sectioning. Another possibility is that your blade needs cleaning and/or sharpening or both. These suggestions may also address the second part of your question.
I agree with you Sheldon R Gordon I have once faced this problem, but it was resolved by keeping the brain for 30 minutes in the cryomatrix solution of Thermo (embedding medium that I have used) at RT before letting it freeze.
Sheldon R Gordon By completely infiltrated, do you mean there could be an interface between the OCT (the embedding medium we use) and the brain? How would I resolve this? I usually pat the surface of the brain with a KimWipe to make sure there is not a layer of sucrose in between OCT and tissue.
I used a fresh blade the day that these photos were taken. I saw the same results when I used an older blade in my previous attempts at cryosectioning.
Liku Biswal A colleague of mine mentioned this, so I let the brains sit in OCT 10 minutes before freezing in the mold--from your experience, is there a chance that I just didn't let it sit long enough before freezing?
Anytime you section any tissue, be it in paraffin, OCT or epon there needs to be a uniformity in the hardness of the sample and the embedding media. This will not be the case if the sample hasn't sat long enough in the embedding medium. The result is that the tissue breaks up as the blade passes through. It because it has not been properly infiltrated with the embedding material. Maybe you need to go though a concentrstion series of OCT (i.e., 10, 25. 50, 75 and finally 100%) to ensure it fully permeates jnto the tissue.
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