David, Two ideas:
My suspician is that this might not be ice-crystal artifact. I have seen similar if the frozen tissue has been stored at -20oC instead of -70oC. What happens is the tissue begins to dessicate (like sausage stored uncovered in the fridge). There was a recent article in the Journal of Histotechnology that might save the day: Iren Horkayne-Szakaly; Glenn D. Sandberg; Joren Keylock; Elisabeth J. Rushing (2009) "Nonfrozen Transport Medium Preserves and Restores Skeletal Muscle Enzymatic Activity and Morphology" J Histotechnol 32(2):49-53 Though I notice that you perfuse with a cold sodium solution. Again since saline is not isotonic with cells, water tends to be dragged into the cells in order to equalise the salt concentrations, causing the cells to swell. When the tissue is frozen, large ice crystals result. See: Henwood, A., (2007) "Adverse effect of saline on brain intraoperative (frozen section) Histology" J Histotechnol 30(3):193. Regards Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC) Laboratory Manager & Senior Scientist Tel: 612 9845 3306 Fax: 612 9845 3318 the children's hospital at westmead Cnr Hawkesbury Road and Hainsworth Street, Westmead Locked Bag 4001, Westmead NSW 2145, AUSTRALIA -----Original Message----- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of David Santer Sent: Friday, 26 March 2010 6:26 PM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] freezing mouse heart tissue Hello, I am currently trying to produce cryosections from mouse heart tissue. I already have experience with paraffin-sections and had faced no major problems. But with cryosectioning I would ask for your help. You can get an idea of our current status at <http://www.d-cup.at/histo/mouseheart.jpg> this link (Hematoxylin test stain, not H&E, the whole heart section looks like this) and as you might guess I am not satisfied with the quality. Would you call this freezing artifacts? Some people suggested that freezing with only LN2 would be not quick enough and create those ice crystals. Here is how we prepared the tissue: After taking out the hearts from the mice, we flushed them retrogradely via the aorta with cold sodium solution. Then we cut the hearts in half, put them into cryomolds and covered them with OCT. Afterwards they were snap-frozen in liquid nitrogen and stored at -80°C. Do you have an advice or maybe a suitable protocol for me? Would you recommend 2-methyl butane? Thank you very much! Greetings from the sunny Vienna! David -- Mit freundlichen Grüßen with kind regards Dr. David Santer Ludwig Boltzmann Cluster for Cardiovascular Research c/o Core Unit for Biomedical Research Waehringer Guertel 18-20 - Leitstelle 1Q A-1090 Vienna Austria Website: <http://www.cardiovascular-research.at> www.cardiovascular-research.at _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ********************************************************************************* This email and any files transmitted with it are confidential and intended solely for the use of the individual or entity to whom they are addressed. If you are not the intended recipient, please delete it and notify the sender. Views expressed in this message and any attachments are those of the individual sender, and are not necessarily the views of The Children's Hospital at Westmead This note also confirms that this email message has been virus scanned and although no computer viruses were detected, The Childrens Hospital at Westmead accepts no liability for any consequential damage resulting from email containing computer viruses. ********************************************************************************* _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
