Immunocytochemistry Using ATTO-Conjugated Primary Antibodies on Fixed & Permeabilized Cells

Cell preparation:

1. Plate cells in chosen chamber slides and grow cells 1-2 days in appropriate medium. Cells need to attach strongly to plate.
IMPORTANT! Some cell lines will need special coating, i.e. poly-lysine of the chamber slides to aid in cell attachment. The specific type of coating needs to be determined empirically as it varies between chamber types and/or cell lines.

2. Wash cells 2-3 times with ice-cold PBS.
3. Fix cells by adding paraformaldehyde (PFA) 1-4% in PBS 10 min at room temperature.
Note: the optimal PFA dilution depends on the cell type and should be established experimentally.

4. Wash cells 2-3 times with ice-cold PBS.
5. Permeabilize cell membranes by adding saponin assay buffer (PBS + 2% BSA + 0.1% saponin) 10 min at room temperature.
6. Block unspecific binding sites with 5% goat serum in saponin assay buffer for 15 min at room temperature.

Labeling:

7. Add ATTO-conjugated primary antibody at the appropriate dilution in saponin assay buffer and incubate for 1-2 hr at 4°C.
8. Wash 3-5 times with saponin assay buffer and drain well. Add buffer to cover cells and detect using a microscope.