Table 2.
Antibodies and conjugates used in the different staining steps for the identification of macrophage/monocytic cells and viability (staining 1), detection of surface expressed viral antigens (staining 2) and presence of MHC I (staining 3) on isolated FIPV positive cells
| Different staining steps (1–6) | Staining 1 | Staining 2 | Staining 3 |
|---|---|---|---|
| 1. Viability staining | EMAa,b | – | EMAa,b |
| 2. Fixation | Formaldehyde 1% | Formaldehyde 1% | Formaldehyde 1% |
| 3. Cell surface staining | Monocyte marker (DH59B) + goat anti-mouse-FITCb | Biotinylated anti-FIPV Ab + streptavidin–Texas Redb | MHC I marker (CF298A) + goat anti-mouse-FITCb |
| 4. Permeabilization | Triton X-100c 0.1% | Triton X-100c 0.1% | Triton X-100c 0.1% |
| 5. Cytoplasmic staining | Biotinylated anti-FIPV Ab + streptavidin–Alexa fluor® 350b (blue) | Anti-FIPV-FITC Ab | Biotinylated anti-FIPV Ab + streptavidin–Alexa fluor® 350b (blue) |
| 6. DNA staining | – | Hoechst 33342b | – |
a
Ethidium mono-azide bromide which specifically stains the nuclei of dead cells (red).
b
Molecular Probes (Eugene, Oregon, USA).
c
Sigma–Aldrich GmbH (Steinheim, Germany).