Materials and reagents

Biological materials

1. Cultured cells: A cell model derived from the H4 neuroglioma line (H4/αS-HoTag3) was used. This model generates α-synuclein (αS) inclusions through stable expression of recombinant αS fused to HoTag3 at the C-terminus, making it ideal for demonstration of CLEM application in cell samples to reveal ultrastructural details of protein aggregates [20]. This cell model is available from the author’s laboratory upon request.

2. Human brain tissues: Post-mortem brain tissues from patients with confirmed neurodegenerative diseases were obtained from the Brain Bank for Neurodegenerative Disorders at Mayo Clinic Florida, with consent and institutional approval.

Reagents

1. Dimethyl sulfoxide (DMSO) (Fisher BioReagents, catalog number: BP231-1)

2. LR white (medium grade) (Electron Microscopy Sciences, catalog number: 14381)

3. Sodium meta-periodate (Thermo Fisher, catalog number: 20504)

4. VECTASHIELD^® antifade mounting medium with DAPI (Vector Laboratories, catalog number: H-1200-10)

5. Toluidine blue O (VWR, catalog number: IC15264925)

6. Sodium borate 10-hydrate (Na₂B₄O₇·10H₂O) (The Science Company, catalog number: NC-1686)

7. Normal goat serum (Thermo Fisher, catalog number: 10000C)

8. Rabbit polyclonal antibody against α-synuclein (Mayo Clinic, catalog number: NACP98)

9. Goat anti-rabbit IgG (H+L) secondary antibody, Alexa Fluor^TM 488 (Thermo Fisher, catalog number: A-11034)

10. Gold-conjugated goat anti-rabbit IgG (H+L) secondary antibodies (12 nm) (Jackson Immuno Research, catalog number: 111-205-144)

11. Sodium cacodylate buffer, 0.4 M, pH 7.2 (Electron Microscopy Sciences, catalog number: 11655)

12. Glutaraldehyde, 10% in H₂O (Electron Microscopy Sciences, catalog number: 16120)

13. Paraformaldehyde 16% aqueous solution, EM grade (Electron Microscopy Sciences, catalog number: 15710)

14. 2% osmium tetroxide aqueous solution (OsO₄) (Electron Microscopy Sciences, catalog number: 19192)

15. TBS with Tween^TM (TBST) (Thermo Scientific Chemicals, catalog number: J77500.K2)

16. 4% uranyl acetate solution (Electron Microscopy Sciences, catalog number: 22400-4)

17. Lead citrate, 3% solution (Electron Microscopy Sciences, catalog number: 22410-01)

18. Gelatin solution 0.1% (Lifeline Cell Technology, catalog number: 102970-804)

19. Sudan Black B Stain kit (ENG Scientific, catalog number: 4460)

Caution: Sodium cacodylate, glutaraldehyde, osmium tetroxide, uranyl acetate, lead citrate, and LR white are toxic chemicals. Always follow standard safety practices to handle and discard these substances.

Solutions

1. 1% toluidine blue in 1% borate (see Recipes)

2. Fixation solution (see Recipes)

3. 1% OsO₄ (see Recipes)

4. 2% uranyl acetate solution (see Recipes)

Recipes

1. 1% toluidine blue in 1% borate

Dissolve 1 g of Na₂B₄O₇·10H₂O in approximately 80 mL of distilled water in a beaker. Add 1 g of toluidine blue O into the borate solution while stirring. Stir the solution until completely dissolved. Filter the solution through a paper filter (Whatman, 1001-918) if particulates remain. Adjust the pH to make sure it is around 9–10, which is suitable for staining. Add distilled water to bring the final volume to 100 mL. Store at room temperature. Label with date and concentration.

2. Fixation solution

Add 0.25 mL of 10% glutaraldehyde, 12.5 mL of 16% paraformaldehyde, and 12.5 mL of 0.4 M sodium cacodylate buffer into 24.5 mL of ultrapure water in a 50 mL high-clarity conical centrifuge tube. Adjust the final volume to 50 mL and mix thoroughly. Store at 4 °C. Avoid light or cover with aluminum foil.

3. 1% OsO₄

Add 5 mL of 2% OsO₄ into 5 mL of ultrapure water and mix completely. Always prepare freshly.

4. 2% uranyl acetate solution

Add 5 mL of 4% uranyl acetate solution into 5 mL of ultrapure water and mix completely. Store at room temperature. Avoid light or cover with aluminum foil.

Laboratory supplies

1. General lab consumables: Gloves, pipettes, mask, paper towel, microcentrifuge tubes, plastic beakers, parafilm

2. Filter paper (Whatman, catalog numbers: 1002-090 and 1001-918)

3. Plastic Petri dish, 100 mm × 25 mm (Sigma, catalog number: Z358762)

4. BEEM^® capsules (Polysciences, catalog number: 00224-500, size 00)

5. Lab microscope slides (Fisher Scientific, catalog number: 12-550-107)

6. 50 mL high-clarity conical centrifuge tubes (Corning, catalog number: 352070)

7. Dry ice

8. Formvar support film single hole 1,500 μm Cu grids (Electron Microscopy Sciences, catalog number: FFGA1500-Cu-50)

9. Formvar support film single hole 1,500 μm nickel grids (Electron Microscopy Sciences, catalog number: FFGA1500-Ni-50)

10. Coverslip forceps (Dumont, catalog number: 11251-33, #5/45)

11. Single edge blade (Uline, catalog number: H-595B)

12. Perfect handle & loop set (Electron Microscopy Sciences, catalog number: 70944)

13. Diamond knife ultra 45 °C, 2 mm (DIATOME, catalog number: DU4520)

14. 100-capacity grid storage box (Electron Microscopy Sciences)

Software and datasets

1. PhotoPad Image Editor (NCH Software)

2. Adobe Photoshop (Adobe)

Equipment

1. Benchtop centrifuge (Fisher Scientific, catalog number: 13-100-678, model: accuSpin 21R)

2. Vacuum oven (Fisher Scientific, catalog number: 13-262-50, Isotemp Model 281A Vacuum Oven)

3. Ultramicrotome (Leica Microsystems, model: Leica UC7)

4. Light microscope (Olympus, model: BX50)

5. Confocal laser-scanning microscope (ZEISS Microscopy, model: LSM 880)

6. Electron microscope (JEOL, model: JEM-1400 FLASH)

7. PELCO^® UVC2 Cryo Chamber (Ted Pella, catalog number: 6202)

8. Rotator (Benchmark Roto-Mini Rotator, model: R2020)

Procedure