1. Grow cells overnight on a 15mm glass cover slide in a 12-well plate in a humidified atmosphere containing 5% (v/v) CO2 at 37°C.
2. Optional: For sphingolipid ExM of bacteria infect the cells according to standard protocols and follow the protocol after bacterial propagation.
Note: Ceramides may exhibit toxic effects on some bacterial species.
3. Exchange the media of the cells for fresh media prewarmed to 37°C
4. Treat the cells with 10 μM α-NH2-ω-N3-C6-ceramide prewarmed to 37°C for 1 hour at 37°C in the exchanged medium
5. Wash the sample with 1x PBS
6. Fix the cells using 4% PFA and 0.1% GA for 10 minutes
7. Wash the sample 3 times with 1x PBS
8. Permeabilize the sample for 15 minutes in 0.2% Triton X-100 in 1x PBS
9. Wash the sample 3 times with 1xPBS
10. Exchange the PBS for 1x HBSS prewarmed to 37°C
11. Incubate the sample in 5 μM Click-IT Alexa Fluor® 488 DIBO alkyne dye at 37°C for 30 minutes. Note: Other functionalized dyes can be used according to preferences.
12. Wash the sample 3 times with 1x PBS
For samples that need additional immunolabelling, e.g. for colocalization of cellular or bacterial proteins, follow the steps a-e before proceeding with step 13.
a. Block the sample with 2% FBS in 1x PBS for 1 hour.
b. Incubate the sample in the primary antibody diluted in blocking buffer for 1 hour in a humid chamber
c. Wash the sample 3 times with 1x PBS
d. Incubate the sample in the corresponding secondary antibody diluted in blocking buffer for 1 hour in a humid chamber
Note: Not all secondary antibodies are suitable for expansion microscopy. Our preferred secondary antibodies are Alexa Fluor 488, CF568, Atto643 and Atto647N.
e. Wash the sample 3 times with 1x PBS
13. Incubate the labelled sample with 0.25% GA at RT for 10 minutes.
14. Wash the sample 3 times with 1x PBS
Note: The monomer solutions for 4x and 10x Expansion are different.
15. For 4x Expansion, add 0.2% ammonium persulfate (APS) and 0.2% tetramethylethylenediamine (TEMED) to the 4x monomer solution
16. Immediately pipet 80 μl of the mixture on parafilm and turn the labeled 15 mm glass slide with the cells facing the bottom onto the drop
17. Incubate for 1 hour at RT for gelation
18. For 10x Expansion, degas 1 ml of the 10x monomer solution on ice for 45 minutes with nitrogen.
19. Add 100 μl KPS (0.036 g/ml) to the monomer solution
20. Degas on ice for another 15 minutes with nitrogen
21. Add 4 μl of TEMED
22. Immediately pipet 80 μl of the mixture on parafilm and turn the labeled 15 mm glass slide with the cells facing the bottom onto the drop
23. Incubate for 30 minutes at RT for gelation, followed by 1.5 hours at 37°C
24. After gelation, remove the gel from the glass cover slide and digest the samples between 3 hours and overnight with 8U/ml protease K in digestion buffer
Note: The cell wall digestion of some bacteria requires additional treatment, e.g.1mg/ml lysozyme in the case of Neisseria gonorrhoeae.
25. Coat glass chambers with PDL overnight at RT.
26. Incubate digested gels in hourly changed ddH2O for expansion until expansion is saturated
Note: Due to the swelling of the gel, make sure that the fully expanded gel has enough space in the dish used, e.g. TC dish 150.
27. Chop expanded gels in pieces fitting the PDL-coated glass chambers for imaging