Methods and Techniques Discussion => General Histology => Topic started by: HPetrie on May 20, 2016, 12:51:41 PM

Title: cryosectioning cystic tissue (mouse)
Post by: HPetrie on May 20, 2016, 12:51:41 PM
Hi all:

Wondering if anyone has some experience that might give insights into my problem. We have a mouse gene mutant that results in the replacement of healthy thymus tissue with cysts.  I assume they are fluid filled, based on the morphology of the epithelial lining, which a simple (probably squamous) epithelial layer.  There is sometimes an included islet of “normal” tissue that is very small. The overall size of these cysts is on the order of 1-2mm. Since the thymus has two lobes, there are two cysts. There is some peripheral accumulation of unilocular adipocytes and fat; most of this is removed, but some remains, possibly equivalent to 10-50% of the cystic/solid tissue at most.

Here is my problem. I can cut 5um fresh frozen sections (in OCT) with no problem; the cysts are either completely intact, or intact enough to be easily recognized, in at least 80% of the sections I cut. However, when I try to fix intact tissue in formaldehyde followed by cryosectioning (this is for analysis of a fluorescent reporter protein), I find it very difficult to cut sections, even at 20um. I have tried a variety of approaches. Of course, I start out with graded sucrose in PBS (final concentration 30%, 48 hours total). I have also tried extensive infiltration (after graded sucrose) with 30% sucrose/50% OCT, I’ve tried changing the blade angle, temperature, etc., everything that I can think of, but still the sections crumple on the blade when there is tissue present. If it weren’t for the fact that I can cut the fresh frozen tissue with no problem, I would think these cysts were just too fragile for cyrosectioning, but that’s obviously not the case.

Not sure what else to try, I am considering sucrose/BSA infiltration to try to fill the cysts with protein, after that I am out of ideas, and I am hoping someone else might have some. Just for the record, paraffin is not an option.

Thanks for your help!