I have a trouble to carry out cerebellar slices in the cryostat. I have samples embedded in tissuetek and I'm cutting slices of 5 to 20 micrometers. However, the majority of slices has many holes. This is a trouble because I need to detect an a injury (with a tincion process, in the myelin), but if all whole sample its full of this holes I cant detect anything, in fact is like if all cerebellum has injury. The blades are new, I use 25-30° for cut and I use an antiroller (I haven't paraffin, then I use tissuetek). At beggining (one month ago aprox) the slices were fine, I mean, the cutting process was the same and the results are fine, but now I have this situation. Somebody can help me and giveme tips?
Hi Abraham, there are several reasons why you are getting slices of poor quality. Firstly, check and make sure that your solutions are fresh and peoperly prepared (concentration and pH). Both, old PFA and wrong molarity of PBS may screw-up the quality of your slices. Secondly, make sure that the perfusion rate is not too fast. Check the literature and make adjustments according to the animals you use. Third, post-fixation i.e. keeping too long in PFA and poor dehydration prior to slicing can deteriorate the quality of your sections. The presence of water in frozen brain tissue can tear the tissue on parts. It is important therefore that the material is kept in sucrose until it floats to the surface. Finally, adjust the temperature of the cryo slicer as over-freezing the tissue may cause its breakage. If you follow these steps, the holes should vanish as they came. Best luck with your slices and let me know how you progress, SV
reach me at [email protected] with your full name , institute name and country with a full query and i will guide you though these issues for solution
The problem may result from tissue being kept in the preservative for too long before sectioning.
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