Table 1.
Antigen retrieval and antibody experimental used conditions at immunohistochemistry.
| Antigen Retrieval | Primary Antibody | Secondary Antibody |
|---|---|---|
| Not performed | Chicken Anti-OPNc IgY; | Goat Anti-Chicken IgY (H + L) Biotin Conjugated |
| Ref # AhOPN-c NA, Gallus Immunoyech INc; | REF #A16058, NOVEX–LIFE TECHNOLOGIES®, | |
| Diluted 1:900 in antibody diluent Solution (Thermo Scientific Quanto, | Diluted 1:3000 in antibody diluent Solution (Thermo Scientific Quanto, | |
| Ref #TA-125-ADQ) for 30 min at RT | REF.# TA-125-ADQ) for 16 min | |
| Antibody retrieval solution (Novocastra™-Leica Biosystems, Ref # RE 7119) 40 min at the steamer cooker, followed by 30 min at RT |
Goat anti- E-cadherin | Biotinylated Goat Polyvalent |
| Ref#3195 Cell Signaling) | (Ref # TP 125-BNE, Thermo Scientific, | |
| Diluted 1:100 Dilution1:900 in antibody diluent Solution (Thermo Scientific Quanto, Ref # TA-125-ADQ) | Solution ready to use, 10 min, RT | |
| for 1 h at RT | ||
| Antibody retrieval solution (Novocastra™-Leica Biosystems, Ref # RE 7119) 40 min at the steamer cooker, followed by 30 min at RT |
Monoclonal mouse anti-Vimentin | Biotinylated Goat Polyvalent |
| Ref # V6389, SIGMA-ALDRICH® | Ref # TP 125-BNE, Thermo Scientific | |
| Diluted 1:400 in antibody diluent Solution (Thermo Scientific Quanto, | ||
| REF.# TA-125-ADQ) for 1h at RT | Solution ready to use, 10 min at RT | |
| Citrate Buffer pH = 6 at microwave oven for 10 min, then 30 min at RT |
Goat anti-total OPN; | Rabbit anti-goat |
| Ref # AF1433, R&D Systems; | Ref # E0466, Dako | |
| Diluted 1:450 in antibody diluent Solution Thermo Scientific Quanto, REF. TA-125-ADQ) for 16–18 h at 4 °C | Diluted 1:200 in antibody diluent Solution (Thermo Scientific Quanto, REF. TA-125-ADQ) for 16 min |
OPN-c: osteopontin-c; Ig: immunoglobulin; ADQ: antibody diluent OP Quanto; RT: room temperature.