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Squash-preparation cytology - (Jun/23/2009 )

Iīm working with Dorosphila melanogaster and I make cytologycal preparation from brains. I have problems to squash them corectly in order to be in one layer so I can easily take photo of mitotic cells. I tried to influence them with trypsin for few minutes to break cell adherent proteins. I got cells in one layer but there were no mitotic cells. I didnīt find any protocol where people used trypsin like this. Do you have any idea where are all the mitotic cells? Or can you give me any tips and triks for cytological squash preparation?


I don't know about insects, but in mammals brains are largely static with few cell divisions occurring once the individual has matured... could this be the case in Drosophila?


Thanks but I donīt think so. I forgot to mention that i used third instar larvae. I have made a lot of samples with DAPI and orcein without trypsin. I saw cells in mitosis but the cells were not in one layer, after trypsinisation they were, but I didnīt see any cells in mitosis. I have a theory that the trypsin do this, but logicaly how trypsin gets into undamaged cell through memmbrane and damage chromosomes in mitosis? This is unanswered question.