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Multiplex staining Using Primary Antibodies Raised in the Same Host

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 two rabbit-raised polyclonal primary antibodies for multiplex staining studies on same floating tissue sections. For this protocol, one antibody must be conjugated to a fluorophore (e.g. ATTO dye).

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. Rinse sections with PBS 2 x 5 minutes.
4. Incubate sections with rabbit primary antibody (un-conjugated) diluted in a solution containing 2% normal goat serum, 0.3% Triton X-100*, and 0.05% Tween-20, for 1 h at room temperature. Transfer to 4°C overnight.
5. Rinse sections with PBS (containing 2% normal goat serum) 2 x 5 minutes.
6. Incubate sections with the secondary antibody, goat anti-rabbit conjugated to a fluorescent dye, in a solution containing 2% normal goat serum, 0.3% Triton X-100*, and 0.05% Tween-20, for 1 h at room temperature. Transfer to 4°C overnight.
7. Rinse sections with PBS (containing 2% normal goat serum) 2 x 5 minutes.
8. Incubate sections with 2% normal rabbit serum (to saturate residual binding ability of the secondary goat anti-rabbit antibody) for 1 h at room temperature.
9. Rinse sections with PBS (containing 2% normal goat serum) 2 x 5 minutes.
10. Incubate sections with rabbit primary antibody conjugated to a fluorescent dye, diluted 1:50-1:60 in a solution containing 2% normal goat serum, 0.3% Triton X-100, and 0.05% Tween-20, for 1 h at room temperature. Transfer to 4°C overnight.
11. Rinse sections with PBS (containing 2% normal goat serum) 2 x 5 minutes.
12. Mount sections on slides and dry for 2 h to overnight.
13. Stain mounted sections with DAPI Nissl stain (blue fluorescence) to label all cells in the field.
14. 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.