Table 1.
Fixation methods and antibodies for immunofluorescent staining
| Tissue | Protein | Fixationa | Primary antibodies | Secondary antibodiesb | Primary antibodies | Zenon-IgG2ac |
|---|---|---|---|---|---|---|
| Body wall muscle | Actin Ce-kettin MyoA | A | Rabbit anti-actin (AAN01), mouse anti-MyoA (5-6) | Cy3-DAR, A647-GAM | MH44 | A488 |
| Actin Ce-kettin Vinculin | A | Rabbit anti-actin (AAN01), mouse anti-vinculin (MH24) | Cy3-DAR, A647-GAM | MH44 | A488 | |
| Actin Ce-kettin Tropomyosin | B | Guinea pig anti-CeTM, mouse anti-actin (C4) | Cy3-DAM, A488-GAG | MH44 | A647 | |
| Ovary | Actin Ce-kettin Vinculin | A | Rabbit anti-actin (AAN01), mouse anti-vinculin (MH24) | Cy3-DAR, A647-GAM | MH44 | A488 |
| Actin Ce-kettin Tropomyosin | B | Rabbit anti-actin (AAN01) MH44, guinea pig anti-CeTM | Cy3-DAR, A647-GAM, A488-GAG | NA | NA | |
| Embryo | Actin Ce-kettin | A | Rabbit anti-actin (AAN01) MH44 | Cy3-DAR, A488-GAM | NA | NA |
NA, not applicable.
a Fixation methods: A, methanol (5 min; −20°C); and B, 4% paraformaldehyde in cytoskeleton buffer (138 mM KCl, 3 mM MgCl2, 2 mM EGTA, and 10 mM MES-KOH, pH 6.1) containing 0.32 M sucrose (30 min at room temperature), followed by methanol (5 min; −20°C).
b A488-GAG, Alexa488-labeled goat anti-guinea pig IgG (Invitrogen); A488-GAM, Alexa488-labeled goat anti-mouse IgG (Invitrogen); A647-GAM, Alexa647-labeled goat anti-mouse IgG (Invitrogen); Cy3-DAM, Cy3-labeled donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA); Cy3-DAR, Cy3-labeled donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories).
c Zenon Alexa Fluor mouse IgG2a labeling reagents. A488, Alexa488 labeled; A647, Alexa647 labeled.