[Cytometry] Protocol conversion to flow

Katie Hickey hickeyk at uoguelph.ca
Fri Jul 10 11:50:15 EDT 2009



I work with bovine and porcine sperm cells and am currently attempting to convert our slide-based immunofluorescent protein localization protocol into a flow (tube-based) protocol.  There are obviously several minor issues which we have worked out however we are struggling with cell permeabilization methods.  We currently keep a set of cells intact and permeabilize the others by dipping the slide in -20 degree methanol for 30 seconds.  Methanol exposure in the tube-based assay is much longer and is deteriorating our very fragile antibody binding sites.  



There are many papers suggesting a myriad of permeabilization methods but I'm wondering if there is anyone frequently doing this type of conversion of protocols that can offer some advice.  



I've tried triton permeabilization with limited success and am aiming to try saponin shortly.  Another consideration is that our protein of interest is located in the very outer plasma mambrane of sperm cells, which is a fragile and dymanic membrane ... seems that everything (temperature, fixation, permeabilization, capacitation status and certainly vortexing) drastically affects our ability to visualize the protein. 



Thanks in anticipation! 


Katie 

Katie Hickey MSc. 
Research Technician 
Animal and Poultry Science 
ANNU 133 
University of Guelph 
Guelph, ON N1G 2W1 

hickeyk at uoguelph.ca 
519-824-4120 x 58355 



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