Hello everyone,
I have been preparing cryosectioned tissue samples of mouse and rat spleens and tumors. I followed the advice of some researchers from here to fix as soon as the tissue was extracted from the animal, and then cryoprotect in 15% and 30% sucrose solutions in 1X PBS until they sunk before finally embedding in OCT medium. Cutting samples prepared in this way has produced very nice samples that do not crack while being cut.
A new problem that I have noticed is that as I am producing these samples, after I collect them from the cryomicrotome and adhere them to the slides, they are cracking before I get a chance to move to the next step of my procedure. I am doing H&E with these samples. I usually leave them to dry a little bit before doing the H&E because I notice that the section adhesion to the glass slide is relatively better if I give it the time to dry. I am using gelatin coated slides with chromium (III) potassium sulfate dodecahydrate (0.2% w/v). This helps the tissue adhere to plain glass slides which otherwise would just fall off.
After just a short amount of time of drying, I see cracks forming and ruining what would appear to perfectly good samples. I have tried fixing the samples again in 4% paraformaldehyde, but this produced really ugly looking samples. I also tried keeping them submerged in a 30% sucrose solution but this made the sample look mushy. I also tried putting it in an cell incubator. This one looked pretty good but still had some cracking. I hadn't got a change to compare it to a slice dried normally so I don't know the difference yet.
Is it a numbers game trying to get a perfect slice, or is there some way to get nearly perfect slices?
Dear Sebastian,
I am NOT an expert in this field. However, I found the attached publication to be important for solving your problem with the cracking samples.
Hoping this will be helpful,
Rafik
Dear Sebastian,
improving your fixation may improve the quality of your tissues. Instead of just dissecting the tissues and placing them in fixatives, which takes a long time for the fixative to penetrate the tissue. I recommend that you fix your animals first by intracardic perfusion, which clears all the blood cells as well as give a superior quality tissue. For details of this method you can see my PhD thesis available here on RG, I did lots of it.
Best wishes, Refik
Dear Sebastian Freeman:
I agree to Refik, fixing the tissue via intracardic perfusion will get good quality of your slices really.
BTW, I just want to provide you another information.
I got the same problem before, but that project had been closed before I figured out the crux. The situation was that I repeated the same protocol from other cooperated lab (they got really pretty outcomes), except the tissue slides they used due to a short sale. I use the substitute (different coating material), and I got really bad looking slices due to cracking. Somehow, the coating might be another factor?
Dear Sebastien. The type of fixative also influence your results. Which fixative are you using?. I agree that perfusion fixation is ideal but not always feasible depending of the study particularly if there is need of simultaneously collecting fresh tissue for other tests. Processing for paraffin embedding still gives much higher histological resolution than cryostat preparations, even in non-perfused animals. Enclosed is one of our publications with methodology for lymphoid tissues. The same procedure can be used for all internal organs. If your lab does not have the facilities contact your nearest pathology lab and they may be willing to assist you or collaborate processing the tissues on you behave. It is a service that pathology labs may be willing to render for a fee. If not you may be able to do it by hand. Enclose one of our publication that may help. Let me know if you need a protocol for perfusion.
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