Materials and reagents

Biological materials

1. Arabidopsis thaliana transgenic fluorescent line pUBQ10::mCherry-MEMB12 and p35S::NAG-EGFP [9,10] or any relevant fluorescent transgenic line

Reagents

1. Distilled water

2. Murashige and Skoog medium (MS) (Duchefa Biochemie, catalog number: M0222.0050)

3. Sucrose (Sigma-Aldrich, catalog number: H4034-500G)

4. Plant agar (Duchefa Biochemie, catalog number: P1001.1000)

5. MES [2-(N-morpholino)ethanesulfonic acid] (Euromedex, catalog number: EU0033-A)

6. 1,4-piperazinediethanesulfonic acid (PIPES) (Sigma-Aldrich, catalog number: P6757-100G)

7. Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) (Sigma-Aldrich, catalog number: E4378-100G)

8. Magnesium sulfate (MgSO₄) (Euromedex, catalog number: P027-A)

9. Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: P5958-500G)

10. Paraformaldehyde (PFA) 16% (Ted Pella, catalog number: 18505)

11. Driselase (Sigma-Aldrich, catalog number: D9515-5G); store at -20 °C

12. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D8418-250ML)

13. IGEPAL CA-630 (Sigma-Aldrich, catalog number: I3021)

14. Normal donkey serum (NDS) (Abcam, catalog number: ab7475-10mL)

15. ChromoTek GFPboosterATTO647N (Proteintech, catalog number: gba647n)

16. ChromoTek RFPboosterATTO594 (Proteintech, catalog number: rba594)

17. FluoTag^®-X4 anti-RFP abberior STAR 635P (Nanotag, catalog number: N0404-Ab635P-L)

18. Anti-MEMB (not commercially available [10])

19. Goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor^TM 594 (Thermo Fisher Scientific, catalog number: A-11012)

20. Tris base (Sigma-Aldrich, catalog number: T6066)

21. Hydrochloric acid (HCl) 37% (Sigma-Aldrich, catalog number: 320331-2.5L)

22. Glycerol (Sigma-Aldrich, catalog number: G5516)

23. Mowiol 4-88 (Merck, catalog number: 475904)

24. 1.4-Diazabicyclo-2.2.29-octan (DABCO) (Sigma-Aldrich, catalog number: 27802)

Solutions

1. MES 0.5% pH 5.8 (see Recipes)

2. 1/2 Murashige and Skoog agar plate (1/2 MS agar plate) (see Recipes)

3. Microtubule Stabilizing Buffer 4/3 solution (MTSB 4/3 solution) (see Recipes)

4. Microtubule Stabilizing Buffer 1× solution (MTSB 1× solution) (see Recipes)

5. Fixation solution (see Recipes)

6. Cell wall digestion buffer (see Recipes)

7. Permeabilization buffer (see Recipes)

8. Blocking solution (see Recipes)

9. Primary antibody solution (see Recipes)

10. Secondary antibody solution (see Recipes)

11. Nanobody solution (see Recipes)

12. Tris 0.2 M pH 7.4 (see Recipes)

13. Mowiol (see Recipes)

Recipes

1. MES 0.5% pH 5.8

Adjust the pH with KOH 10 N.

Note: The solution can be stored for several months at room temperature.

2. 1/2 MS agar plate

a. Mix the solution.

b. Autoclave for 30 min at 110 °C.

c. Under sterile conditions, add 50 mL of solution per square plate.

d. Let it polymerize for 30 min at room temperature.

Note: The plates can be stored for several weeks at 4 °C in sterile conditions.

3. MTSB 4/3 solution

Adjust pH to 7 with KOH.

Note: The MTSB 4/3 solution can be sterilized (110 °C, 30 min) and stored for several months at 4 °C.

4. MTSB 1× solution

Adjust pH to 7 with KOH.

Note: The MTSB solution can be sterilized (110 °C, 30 min) and stored for several months at 4 °C.

5. Fixation solution

Note: Store PFA at 4 °C. PFA is a carcinogen and needs to be manipulated under a flow hood.

6. Cell wall digestion buffer

Note: This solution needs to be freshly prepared. After resuspension, centrifuge briefly at 500× g at room temperature to pull the debris of the Driselase extract powder.

7. Permeabilization buffer

Note: This solution needs to be freshly prepared.

8. Blocking solution

Note: This solution needs to be freshly prepared.

9. Primary antibody solution

Note: In this study, we used anti-MEMB, but any relevant primary antibody can be used. For other antibodies, we advise testing a range of dilutions from 1/100 to 1/600. This solution needs to be freshly prepared, and a range of dilutions needs to be tested to optimize the immunolabeling.

10. Secondary antibody solution

Note: In this study, we used anti-rabbit secondary antibodies, but, obviously, the secondary antibody needs to target the host species of the relevant primary antibody previously used. This solution needs to be freshly prepared, and a range of dilutions needs to be tested to optimize the immunolabeling.

11. Nanobody solution

Alternative labeling strategies: If a double marker transgenic line is used with GFP- and RFP-tagged (or other fluorescent protein derived from GFP and RFP) proteins, select one of the combinations below:

Note: These dilutions are set up for NAG1-EGFP and MEMB-mCherry; if other fluorescently tagged proteins are used, we advise testing a range of dilutions from 1/200 to 1/2,000. This solution needs to be freshly prepared, and a range of dilutions needs to be tested to optimize the immunolabeling. Several nanobodies can be incubated simultaneously.

12. Tris 0.2 M pH 7.4

Adjust pH to 7.4 with the addition of HCl 37%.

13. Mowiol

a. Shake the solution for 4 h at room temperature on a rotative wheel.

b. Let the solution rest for 2 h at room temperature.

c. Incubate for 10 min at 50 °C in a water bath.

d. Centrifuge at 5,000× g for 15 min.

e. Recover the supernatant.

f. Adjust the pH to 7.4 using HCl 37%.

Note: The use of pH paper is recommended for this step. If the pH level is exceeded by mistake, discard the solution and make it again (see Troubleshooting).

g. Prepare 500 μL aliquots in 1.5 mL Eppendorfs and store at -20 °C for long-term storage.

Laboratory supplies

1. Falcon 50 mL (Sarstedt, catalog number: 62.547.254)

2. 1.5 mL Eppendorf (Sarstedt, catalog number: 72.706.400

3. Square Petri dish 120 × 120 mm (Dutscher, catalog number: 030555S)

4. 6-well plate (SARSTEDT, catalog number: TC-Plate 6 wall, suspension F 83.3920500)

5. Fine dissection clamp (Fisher Scientific, catalog number: 13-812-41)

6. SuperFrost Plus ground edge 90° clipped corner x72 (Labelians, catalog number: LSFPLUSCC)

7. Razor blade (Swann-Morton, catalog number: 0213 No26)

8. Hydrophobic pen (VectorLab ImmEdge, catalog number: H-4000 (ZL0627)

9. Slide box (Merck, catalog number: HS15991A)

10. Coverslip (VWS, catalog number: 631-0125 thickness N°1.5 Borosilicate Glass)

11. Transparent nail polish (Rimmel london, catalog number: 740 clear-8mL)

12. Magnetic stirrer (Thermo Scientific, catalog number: 88880007)

13. Magnetic bar (Thermo Scientific, catalog number: 50087930)

Equipment

1. Autoclave PBI Stematic III (GEMINI, catalog number: 05523)

2. Table shaker (Infors HT, model: Orbitron)

3. Balances (Sartorius)

4. Centrifuge for Falcon tubes 50 (Beckman Coulter, model: Allegra21R)

5. pH meter (Mettler TOLEDO, model: seven compact)

6. Microscopy: The equipment used included a Leica SP8 WLL2 confocal microscope mounted on an inverted DMI6000 stand (Leica Microsystems, Mannheim, Germany), equipped with a HC PL APO CS2 motCORR 93× glycerol objective (NA 1.3). The system featured an 80 MHz pulsed white light laser 2 (WLL2) with tunable excitation from 470 to 670 nm in 1 nm increments. Scanning was performed using a conventional scanner operating at frequencies ranging from 10 to 1,800 Hz. Detection was achieved via two internal photomultiplier tubes (PMTs), two internal hybrid detectors, and one external PMT for transmission. Additionally, the microscope was outfitted with a 3D STED module, enabling STED microscopy in both XY and Z directions. A variable allocator split the laser beam into two paths to shape the light: a two-dimensional (2D) STED doughnut using a vortex phase plate and a three-dimensional (3D) STED z-doughnut using a pi-step phase mask. The STED setup included a 775 nm depletion pulsed laser (80 MHz) and was exclusively used with the 93× glycerol 1.3 objective. The FALCON module allowed for fluorescence lifetime measurement, enhancing STED images through photon filtering by lifetime (tau-STED).

Software and datasets

1. FIJI (ImageJ, Version 1.54p)

2. LASX 3.5.10

Procedure