This tool may help in the design your experiment. It will apply the criteria outlined above to identify the potential cross-reactivity between primary antibodies and therefore the minimal cross-reactivity each secondary antibody will require.
Input the species of your sample and the antibodies you intend to use. It is preferential to use the same host species for all the secondary antibodies to minimise the potential for cross-reactivity. Antigen names can be customised by editing the text directly in the table.
Please note that every potential minimal cross-reactivity permutation may not be available, even if indicated by the tool. Goat and Donkey are typically the best secondary antibody host species choice due to the availability of minimal cross-reactivity options.
Fluorescent probes should have the required color separation, read more about this here.