Immunocytochemistry using 8-well Chamber Slides McAllister Lab, updated 09.21.09

Setup: Plate cells at desired density into wells of 8 well chamber slides (Nalgene Nunc International, cat. no. 177402) and treat directly in wells as required. Wells hold volume of 200-500l. Staining Protocol: 1. Remove medium and wash cells 3x with PBS 2. Fix cells in 4% paraformaldehyde – 10 min on ice 3. Wash cells 3x with PBS 4. If required, permeabilize cells in 100% EtOH – 10 min on ice 5. Wash 3x with PBS 6. Block either with 1% BSA or with serum from species in which secondary Ab is raised (approx. 1 drop serum in 5 ml PBS). Incubate 5 min at 40oC. 7. Wash 3x with PBS 8. Incubate with primary antibody - 20 min at 40oC. 9. Wash 5x with PBS 10. Incubate with appropriately labeled secondary antibody - 15 min at 40oC. 11. Wash 5x with PBS 12. Nuclei counterstain: Hematoxylin (Biomeda, cat. no. M10) 1 min, room temp. OR 1X DAPI (Sigma cat. no. D9542) 15 min, room temp. 13. Wash cells with dH2O 14. Remove gasket and rubber seal from chamber slides; rinse with dH2O 15. Cover samples with mounting media and coverslip. For light microscopy, use Crystal Mount (Biomeda, cat. no. M02). For fluorescence, use anti-fade reagent (Molecular Probes, cat. no. S-2828) . Seal coverslip with nail polish if necessary. Notes: Remove reagents from wells using a clean glass pipette attached to vacuum apparatus. It is important to wash cells thoroughly between incubations. For incubation steps at 40oC, slides can be placed directly onto a heat block equilibrated to target temperature or placed in a warm room.