Protocols for Immunocytochemistry in cell culture

General Protocols for Staining from Members of the Core

There are many small variations in staining, so keep in mind that, as with any protocol, you may need to optimize the below protocols for your needs.

Many companies have resources on staining that may be helpful to check out:

• Abcam: IHC Guide
• Thermo Fisher: Cell and Tissue Analysis Microscopy Protocols

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ICC — Immunofluorescent Staining in Cell Culture

Procedure on pure neuronal primary cell culture — adjust accordingly (done on coverslips in 24-well plates).

Antibodies:

• Primary antibody made in Rabbit / Mouse
• Secondary antibody: Anti-Rabbit / Anti-Mouse

Protocol:

1. Wash cells in 1x PBS — 5 min
2. Fix cells with 4% PFA at RT — 10 min
3. Wash cells with 0.01% PBS-Triton X-100 — 5 min
4. Permeabilize cells with 0.1% PBS-Triton X-100 — 10 min
5. Wash cells with 0.01% PBS-Triton X-100 — 5 min
6. Block cells with 10% Normal Donkey Serum in 0.01% PBS-Triton X-100 — 60 min at RT
7. Incubate with primary antibodies (mouse anti- and rabbit anti-) in 3% Normal Donkey Serum in 0.01% PBS-Triton X-100 — 2–16 hrs at 4°C
8. Wash cells with 0.01% PBS-Triton X-100 — 5 min
9. Incubate with secondary antibodies (anti-mouse and anti-rabbit) in 3% Normal Donkey Serum in 0.01% PBS-Triton X-100 — 2 hrs at RT
10. Wash cells in 1x PBS — 5 min
11. Wash cells in 1x PBS — 5 min
12. Wash cells in 1x PBS — 5 min
13. Mount onto slide

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Staining of Suspension Cells on Coverslips

📄 Fixing and Staining Suspension Cells — Coverslips Protocol

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Staining of Suspension Cells in Ibidi Dishes

📄 Fixing and Staining Suspension Cells — Ibidi Protocol

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