Immunocytochemistry (ICC)

Abstract

This protocol describes how to do immunocytostochemistry for primary and hiPSC-derived cells.

Steps

Cell fixation

Cells are fixed in 4% volume paraformaldehyde (PFA) and stored in phosphate-buffered saline (PBS) until use.

Permeabilizing and blocking cells

Wash cells with PBS twice.

Incubate the cells in 0.2% volume Triton X-100, 5% volume bovine serum albumin (BSA) for 1h 0m 0s atRoom temperature.

Note

5% volume BSA (made in PBS) is used to block non-specific binding.

Note

For ATTO 425 labelled Aptamer staining, cells are permeabilized with 0.25% volume Triton X-100 and blocked with 10% volumenormal goat serum (NGS) for 0h 20m 0s followed by another 3h 0m 0s with 0.1% volume Trion X-100 and 10% volume NGS.

Incubate cells in primary antibodies

Note

Do not wash after permeabilising and blocking steps

Dilute primary antibody in 5% volume BSA and incubate cells at 4°C, 1h 0m 0s or 1h 0m 0s at Room temperature.

Note

The final volume should be sufficient to cover each coverslip around 170µL for 8-ibid chambers, #80806). For 8-ibid, it recommends incubating the cells at room temperature for 1h 0m 0s at Room temperature if possible.

Wash cells with 5% volume BSA for 0h 5m 0s three times.

Incubate cells in secondary antibodies

Dilute primary antibody in 5% volume BSA and incubate cells at 4°C 0h 5m 0s or 1h 0m 0sat Room temperature.

Wash cells with 5% volume BSA for 0h 5m 0s three times away from light.

Note

Add Hoechst (10micromolar (µM)) in the second wash and leave for 0h 15m 0s.

Take away PBS and load anti-fading medium to cover cells.

Note

For the short term, imaging in PBS is also fine.

Abstract

Steps

Cell fixation

Permeabilizing and blocking cells

Incubate cells in primary antibodies

Incubate cells in secondary antibodies

推荐阅读