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

Filipa Pontes de Moraes fmoraes <@t> igc.gulbenkian.pt
Wed Jan 17 10:18:10 CST 2007


I planning to perform a whole mount double immuno-fluorescence for =20
vessels and smooth muscle cells ( 1Ab solution: rat anti-mouse =20
Pecam)  (2nd Ab sol: Goat anti-rat Alexa488 together with Cy3 =20
conjugated mouse antiSMA)  using mouse embryos. I would like to know =20
what could be the maximum time of 2ndAb solution? Usually is o/n at =20
4=FBC What could happen if instead of one night the embryos stayed 2 =20
days in 2nd Ab solution?

What is the maximum time the embryos could stay mounted in the slides =20=

without loosing the signal? I am planning to mount them in methyl =20
salicilate (SIGMA) used as the clearing agent.

I am having problems with this clearing agent Methyl salicilate, it =20
damage my embryos i just add drop by drop and it is still very =20
difficult to avoid damaging the E10.5 embryos. Can you advice me =20
other organic clearing agent? i have seen  1 benzil alcohol : 2 =20
benzil benzoate used as cleaing agent and mounting reagent also is it =20=


Thank You very much



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