Preparation of samples for confocal microscopy
| Materials | |
|---|---|
| Coverslips (round, 12 mm) | |
| 24-well plate | |
| 4% Paraformaldehyde (PFA) in PBS or | |
| ice-cold methanol or acetone (-20°C) in porcelain well plate |
Procedure
Cells:
- One day before seeding the cells: distribute cleaned and sterilized coverslips in a 24-well plate and coat with gelatine-, poly-L-lysine (10 µg/mL), or fibronectin (2 µg/mL) solution in PBS overnight (o/n) at 4°C
- Aspirate coating solution
- Seed the cells in 0.5-1 mL medium: HEK293T cells: 8x104 cells/well fibroblasts: 3x104 cells/well
Bacteria:
- One day before infection, streak bacteria on plate and incubate at 37°C o/n
- Prepare bacteria for infection according to separate protocol
Infection:
- Before infection: investigate cell distribution/attachment and check for contamination
- Infect the cells for desired time, wash, and fix according to separate protocol
Fixation
- At the end of the assay, wash cells 2 x with 500 µl cold PBS++ (alternatively, fix cells directly without washing by carefully sublayering 500 µl of cold PFA- fixation solution
- For PFA-fixation add 300µl 4% PFA/well and fix for 5 min at RT
- For methanol or acetone fixation: -transfer washed coverslips from PBS into porcelain well plate on ice and add directly into ~200 µl fixative/well (ice-cold MeOH or acetone). Fix/permeabilize for 10 min on ice. Transfer coverslip back to PBS-containing well in plastic 24-well plate. Make sure not to switch orientation of the coverslip.