ICC protocol for staining nucleoporins

[Sai101396]

Hello everyone!
I am looking to stain NUPs in mouse podocytes through ICC. Does anyone have a recommended protocol? I have Alexa Fluor Conjugated Abs from Santa Cruz. Please let me know if possible. Thank you, Sai

[radtkea]

Hi Sai,

Thank you for asking your question in the open! I haven't done this before, but I did found a few resources.
Abcam ICC protocol
Kidney ICC protocol

Here's a start:

1. See Abcam article about suspensions or cells on coverslips.
2. Fix with 4% PFA for 20 minutes. Wash 3X in PBS to remove fixative.
3. Permeabilize with 0.1% Triton X in PBS for 5 minutes. (See Abcam article about different detergents).
4. Block with 3% BSA in PBS for 1 hour.
5. Incubate cells with primary antibodies overnight at 4°C. (Would advise you use the same blocking/labeling buffer).
6. Wash 3X in PBS.
7. Optional: Incubate with secondary antibodies for 1 hour.
8. Wash 3X in PBS.
9. Follow counterstain (DAPI) and mounting media steps in your lab.

Best of luck!
Andrea