Aquaporin-5 Antibody:
Immunocytochemistry Protocol
Culture cells to appropriate density in 35 mm culture dishes or 6-well plates.
1. Remove culture medium and add 10% formalin to the dish. Fix at room temperature for 10 minutes.
2. Remove the formalin and rinse with PBS.
3. Remove PBS and add permeabilization solution (i.e. PBS+0.05%Triton-X100). Be sure to not let the specimen dry out. Wash three times for 5 minutes with washing solution (i.e. PBS).
4. To block nonspecific antibody binding incubate in 10% normal goat serum for 1 hour at room temperature.
5. Add primary antibody at appropriate dilution and incubate at room temperature from 1 hour at room temperature to overnight at 4C.
6. Remove primary antibody and replace with washing solution. Wash three times for 5 minutes.
7. Add secondary antibody at appropriate dilution. Incubate for 1 hour at room temperature.
8. Remove antibody and replace with wash solution, washing three times for 5 minutes. Incubate with DAPI at 2ug/ml for 1 minute.
9. Cells can be viewed directly after washing. The plates can also be stored in PBS containing Azide covered in Parafilm (TM). Cells can also be cover-slipped using Fluoromount, with appropriate sealing.
*The above information is only intended as a guide. The researcher should determine what protocol best meets their needs. Please follow safe laboratory procedures.
