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Multiplex Staining Using Primary Antibodies Raised in Different Hosts

Immunohistochemistry (IHC) is based on the immunodetection of target proteins in tissue sections. The majority of protocols available describe immunohistochemistry using one antibody alone. In practice, however, multiple antibodies are commonly used. We describe our IHC protocol using both rabbit-raised and guinea pig-raised polyclonal primary antibodies for multiplex staining studies on same floating tissue sections.

Staining procedure:

1. Rinse floating sections with phosphate buffered saline (PBS) 2 x 5 minutes.
2. For antigen retrieval and to quench endogenous peroxidase activity, incubate with 0.2 % hydrogen peroxide in 0.1 M phosphate buffer pH 7.3 containing 0.2% Triton X-100* and 20% methanol for 25 min at room temperature.
3. Incubate sections with a cocktail containing two primary antibodies: the first raised in rabbit diluted (1:200 to 1:400)  and the second raised in guinea pig diluted (1:200 to 1:400) in a solution containing 2% normal goat serum, 2% normal donkey serum, 0.3% Triton X-100*, and 0.05% Tween-20, for 1 h at room temperature. Transfer to 4°C overnight.
4. Rinse sections with PBS (containing 2% normal goat serum and 2% normal donkey serum) 2 x 5 minutes.
5. Incubate sections with a cocktail of diluted secondary antibodies: anti-rabbit conjugated to a fluorescent dye and anti-guinea pig conjugated to a different fluorescent dye in a solution in a solution containing 2% normal goat serum, 2% normal donkey serum, 0.3% Triton X-100*, and 0.05% Tween-20, for 1 h at room temperature. Transfer to 4°C overnight.
6. Rinse sections with PBS (containing 2% normal serum) 2 x 5 minutes.
7. Mount sections on slides and dry for 2 h to overnight.
8. Stain mounted sections with DAPI Nissl stain (blue fluorescence) to label all cells in the field.
9. Cover-slip slides with Immumount (Shandon, UK).
* If using an “extracellular” antibody decrease Triton X-100 to 0.05%.

Note: this protocol is only a recommendation. Further calibration by the end user may be required accordingly.