In double immunoflourescence experiment by confocal, I intend to use two conjugated anti-cd11b and anti-f4/80 Ab (PE and FITC) in mice liver sections. I need your opinion whether,
1) Conjugated Ab with R-PE could be used with FITC?
2) In case of conjugated Ab, is it possible to use two Ab from same host?
1. Make sure you have either tunable filters or very tight filters on your confocal settings and you excite and capture each channel sequentially. PE is not optimal compared to something like Alexa555 because of its excitation spectra which has second peak identical to FITCs excitation peak.
2. yes
I would like to explain that, for the second question, as you trace different Ags so you can use Abs from the same host. But if course you could not trap the same Ag by Abs from different hosts.
Good luck
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