Click-ExM imaging of Cho-contained phospholipids
Metabolic incorporation of Alk-Cho
1. Prepare 100 mM Alk-Cho stock solution using ddH2O. Then prepare the labeling medium by diluting stock solution into cell culture medium to a final concentration of 100-200 uM.
Note: Other lipid labels are usually dissolved in DMSO, and the labeling medium should be vortexed vigorously before being added to cell cultures.
2. Culture the cells in the labeling medium for 12 h at 37 ℃.
Cell fixation and permeabilization (at room temperature, r.t.)
3. Wash cells with PBS for three times. Fix cells with 3% (w/v) formaldehyde and 0.1% (v/v) glutaraldehyde in PBS for 15 min. Treat cells with 0.1% (w/v) sodium borohydride for 7 min, and wash three times with 100 mM glycine in PBS.
4. Permeabilize cells with 0.1% (w/v) saponin in PBS for 5 min and washed three times with PBS.
Pause point: If necessary, samples could be stored temporarily at 4 ℃, but long-time storage may cause potential signal loss or distortion.
Click labeling with azide-biotin (at r.t.)
5. Prepare 200 uL click reaction mixture as the follows.
188 uL 1×PBS
1 uL azide-PEG3-biotin (10 mM)
1 uL BTTAA/CuSO4 complex (6:1, mol/mol, 10 mM)
10 uL sodium ascorbate (50 mM, freshly prepared, the final step)
6. Incubate cells with the mixture for 1 h with gentle shaking.
7. Wash with PBS for five times.
8. Incubate cells with 5 ug mL-1 streptavidin-dye in PBS containing 1% (w/v) BSA for 1 h, and washed three times with PBS.
Pause point: If necessary, samples could be stored temporarily at 4 ℃, but long-time storage may cause potential signal loss or distortion.
Gelation, digestion and expansion
9. Incubate samples with 0.1 mg mL-1 AcX in PBS at r.t. overnight, or incubate the cells with 0.25% (v/v) GA in PBS for 10 min.
10. Wash samples three times with PBS.
11. Prepare monomer solution as the follows.
1×PBS
2 M NaCl
2.5% (w/v) acrylamide
0.15% (w/v) N,N’-methylenebisacrylamide
8.625% (w/v) sodium acrylate
12. Prepare 1 mL gelation solution as the follows.
940 uL monomer solution
20 uL ddH2O
20 uL 10% (w/w) TEMED (freshly prepared)
20 uL 10% (w/w) APS (freshly prepared, the final step)
13. Incubate samples with the gelation solution at 4 °C for 5 min, and then transfer to a humidified 37 °C incubator for 1 h for gelation.
Note: Before placing the samples in a 37 °C incubator, keep the solutions chilled to 4°C, and avoid warming the gelling solution.
14. Prepare digestion solution as the follows.
50 mM Tris, pH 8.0
1 mM EDTA
0.1% (v/v) Triton X-100
0.8 M guanidine HCl
8 units mL-1 proteinase K (added before use)
15. Digest the hydrogel in digestion buffer at 37 °C for different time. In general, 4 h for AcX-anchored samples and 2 h for GA-anchored samples.
Pause point: If necessary, the gel could be stored in PBS temporarily at 4 ℃, but long-time storage may cause potential signal loss or distortion.
16. Place the hydrogel into ddH2O to expand. Change water every 20 min until expansion was complete.
17. Optional: Stain the nuclei with Hoechst 33342 (5 ug mL-1) to facilitate locating the cells in the hydrogel.
Imaging
18. Coat 24×50 mm rectangular No. 1.5 coverglasses with 0.1 mg mL-1 poly-D-lysine for 10 min at r.t. and allow to dry.
19. Remove excess water carefully using laboratory wipes, and place the expanded samples on the coverglasses.
20. Set up microscope and perform imaging. In general, find the region of interest using low-magnification objectives with long working distance, and then switch to high-magnification objectives.
Note: For ExM procedure, other details could be found in the online methods and previous ExM references. For click-labeling procedure, this protocol is also applicable for other metabolically labeled biomolecules in cultured cells by changing to corresponding reagents. A few notes of click labeling are listed as follows.
1. In some cases, click labeling with alk-biotin/dye might cause higher background staining than azide-biotin/dye, enough washing with suitable surfactant (e.g., Tween 20) could solve this problem.
2. To increase the labeling efficiency of nucleic acids (e.g., metabolically labeled with EdU/EU), consider using Cu/THPTA instead of Cu/BTTAA, and try to scale up the concentration of each components.