Methods and Techniques Discussion > General Histology

white clumps in frozen sections of spleen

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Rams:
Hi everyone,

I posted last week a new topics in "general discussion" regarding a problem which is quite puzzling for me. I receive interesting suggestions and ideas but unfortunately after trying all of them I did not overcome the problem, .
Each time I try to cut sections of spleen from HELATHY and naive mice I observe white clumps forming during sectioning. I do not know whether these clumps are due to formation of ice crystals but they turned into holes after sectioning. I used various protocol to freeze the organs, including slow freezing with dry ice, snap freezing with or without ispoentan and liquid nitrogen but no way to avoid these artefacts to occur. I joined a picture of the spleen if you need more informations.
I would really appreciate your help and ideas, as I need to make urgently some sections from my immunized mice...
Thanks a lot for your help!!!!!
Rams

richard03:
You should cover tissue with OCT before freezing it. If you did freezing before applying OCT, it may form ice crystal and holes in tissue. It is critical.

Rams:
Hi Richard,
I've always frozen my tissue after OCT embedding. This was apparently not sufficient to avoid ice crystals to form, whatever the technique of freezing used was.
I've not yet solved the problem...
Thanks for your reply
Rams

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