Table 3.
Staining for the Three Most Frequently Used Antibodies Directed against Ep-CAM
| Antibody | Staining method | |
|---|---|---|
| Paraffin | Cryostat | |
| 323/A3 | Fixation: methanol (5 minute; Tr) and 0.3% H2O2 (20 minute; Tr) | Fixation: acetone (10 minute; Tr) |
| Pretreatment:- | ||
| Pretreatment: 0.1% trypsin (w/v) in 0.1% CaCl2 (w/v) (20 minute; Tr) | Incubation 1st Ab: 5 μg/ml in PBS/1.0% BSA (1 hour; Tr) | |
| Incubation 1st Ab: 1 to 5 μg/ml (overnight; Tr) | Detection: two-step biotin/streptavidin system or envision | |
| Detection: two-step biotin/streptavidin system | ||
| Ber-EP4 | Fixation: formalin | Fixation: acetone (10 minute; Tr) |
| Pretreatment: 0.1% trypsin (w/v) in 0.1% CaCl2 (w/v) (20 minute; Tr) | Pretreatment: - | |
| Incubation 1st Ab: 6 μg/ml (overnight; Tr) | Incubation 1st Ab: 5 μg/ml (45 minute; 40 °C) | |
| Detection: two-step biotin/streptavidin system | Detection: Vectorstain Elite kit | |
| KS1/4 | Fixation: formalin | Fixation: 4.0% formaldehyde (20 minute; 4 °C) |
| Pretreatment: 0.1% trypsin (w/v) in 0.1% CaCl2 (w/v) (20 minute; Tr) | Permabilization: 0.1% saponin (5 minute; Tr), 50 mM glycine in PBS | |
| Incubation 1st Ab: 5 to 10 μg/ml (overnight; Tr) | Pretreatment: - | |
| Incubation 1st Ab: 5 μg/ml (1 hour; Tr) | ||
| Detection: two-step biotin/streptavidin system | Detection: immunofluorescent labeled 2nd Ab | |
Tr: room temperature.