thanks a lot Dr. @mohamed

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.

@Maria !1 Thank you so much Prof

If both antibodies are same species, another option is to use dye-labeled primary antibodies.

http://www.pnas.org/content/110/29/11982.full.pdf

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.

@Renu Pal Thank you so much Prof.