Hi, I encounter some problems, I need to my research to fix and cryoprotec some blood clot for histology analyses. Mys current protocol is the following : 48h of para formaldehyde 4% and 7 days of sucrose solution 20% (the solution is changed every day). To freeze the clot I used liquid nitrogen and I conserve them at -80°C. To cut them I use a Cryostat at -26°C for the chamber temperature and -18°C for the object (not modifiable). The section thickness are 10µm. When I cut the clot I observe a tissue degradation at the center of the clot. (see picture).
Does anyone have any suggestion to avoid the tissue degradation ?
Thank you in advance.
After cryoprotection step (sucrose treatment), instead of liquid Nitrogen snap freezing in OCT, transfer your sample into OCT containing tube or small container and mix your tissue with OCT by hand-rotating (slowly rotate your tubes) and leave the tubes with OCT + tissue at RT for about 5 min. It will exchange/ or cover up the tissues with OCT. then transfer your samples into OCT containing tissue mold. You can transfer your sample to negative 20 degree to freeze it (if the orientation of tissue in the OCT is not critical), and transfer it to negative 80 next day.
Thank you Enkhtuya Radnaa, in fact my explanation lack some details. I do transfer my object in OCT before freezing. I form a small cube in aluminum foil in which I place my tissue and fill it with OCT. I leave my object like that for about 5 min and I freeze them in liquid nitrogen. I have tried to do It with dry ice it also work but it takes a little bit longer. Usually for brain or other structure I use the same protocol and I don't have the same freeze artifact on my sample.
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