[Histonet] Immuno-fluorescence: maximum time in Ab solution?

From:Filipa Pontes de Moraes


I planning to perform a whole mount double immuno-fluorescence for  
vessels and smooth muscle cells ( 1Ab solution: rat anti-mouse  
Pecam)  (2nd Ab sol: Goat anti-rat Alexa488 together with Cy3  
conjugated mouse antiSMA)  using mouse embryos. I would like to know  
what could be the maximum time of 2ndAb solution? Usually is o/n at  
4ūC What could happen if instead of one night the embryos stayed 2  
days in 2nd Ab solution?

What is the maximum time the embryos could stay mounted in the slides  
without loosing the signal? I am planning to mount them in methyl  
salicilate (SIGMA) used as the clearing agent.

I am having problems with this clearing agent Methyl salicilate, it  
damage my embryos i just add drop by drop and it is still very  
difficult to avoid damaging the E10.5 embryos. Can you advice me  
other organic clearing agent? i have seen  1 benzil alcohol : 2  
benzil benzoate used as cleaing agent and mounting reagent also is it  

Thank You very much



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