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Hi all,I've been trying to cut some cryosections of mouse spleen but I face with a recurring troubleshooting.Whatever the protocol used to freeze the spleen in O.C.T (slow freezing using dry ice, snap freezing with liquid nitrogen in the presence or absence of isopentane etc..) I always see white clumps forming into the organ once cut with the cryostat. I tried to play with the temperature of the cryostat chamber as well as the temperature of the knife but no way to avoid these strange clumps appearing. I've never seen that in the past while I've cut many times this organ and I don't know whether these "holes" are due to the formation of ice crystals, but in the presence of isopentane I should not see them anymore?I joined below a photo of the organ to illustrate the problem.If anyone had any suggestion, protocol or idea to help me solving the problem, I would appreciate a lot!!Thanky ou very muchRams
Hi,First I would like to thank you all for your help and your interesting suggestions.Indeed these white clumps appearing in the spleen turn to be holes after sectioning as a quick H&E staining and a observation under the scope do not reveal any cellularity aspect in these white regions.Moreover, I tried many times to take the spleen from freshly arrived mice, from various species, naive, but the problem persisted. Importantly, these mice do not develop any tumor and do not receive any treatment. Your suggestion Cardio can be of interest. I'll try to put less OCT in the tissue, hoping that the clamps will not appear during the sectioning...Sorry to repeat myself but I tried several methods of freezing without any success for the moment...The problem seems to concern specifically the spleen as other organs like the lung do not show these curious artefacts.I am looking forward to receiving other pertinent ideas from youThank you for your helpRams
Hi,I do not know whether the antibody I am going to use to stain the sections of the spleen (once I will solve the problems of the white clumps) will work on fixed tissues (PFA, formalin) I wonder if you have ever tried to put your spleen in 30% sucrose O/N at 4°C without any previous fixation? Thank youRami