In this case one always uses two separate antibodies against two antigens raised in two different host species i.e. rabbit and mouse or rabbit and goat etc. Note that, for any kind of immunolabelling, optimum primary and secondary antibody concentrations are established by trial and errors by starting from the manufacturer's specifications.
Combine both primary antibodies in one tube at their respective dilutions. Do the same for the secondary antibodies.
EXAMPLE: |
| In order to dual stain using IP3R2 and S100b, mix the primary antibodies as follows: |
| IP3R2 is used at 1:100 , S100b is used at 1:2000 |
| For 1ml, add 10µl of IP3R2 and 0.5µl of s100b |
| Mix the secondary antibodies as follows: |
| IP3R2 was raised in a rabbit - use RRX conjugated Goat anti Rabbit at 1:100 |
| S100b was a mouse monoclonal ab - use FITC conjugated Goat anti mouse at 1:100 |
| For 1ml, use 10µl of each secondary antibody. |
