I am interested to check two antibodies on colon tissue (paraffin embedded) which I strongly believe show co-localization. What is the simple and best protocol ?
Dear Rohit, In case you have your primary antibodies raised in two different species, you can use a simultaneous incubation - please see the link to the protocol below
http://www.abcam.com/ps/pdf/protocols/double%20immunofluorescence%20-simultaneous%20protocol.pdf
Good luck with your project.
Also, directly conjugated primary antibodies are a good choice if the target proteins are in abundance. Unconjugated primaries offer a chice of disclosure methods and a wide range of enhancements, so for many researchers this is a default method. When cost is an issue - the cost per slide, especially for the larger projects, is lower when using unconjugated primary antibodies.
Hello Rohit, It is definitely possible when your primaries are of two different species as Maria mentioned already. For the secondaries you can use Alexa 488 anti-mouse and Alexa 568 anti-rabbit or vice versa to identify them separately when you view through microscope. All the Alexa antibodies give strong signals so you can rely on them with no problem. Good luck.
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