Electron Microscopic Detection of Proteins and Protein Complexes in Mammalian Cells Using APEX-tagged, Conditionally Stable Nanobodies

Materials and Reagents

1. 9.2 cm² (40 mm diameter) tissue culture dishes (TPP, supplied by Sigma-Aldrich, catalog number: 93040)
2. Gloves
3. 0.2 µm syringe filter
4. 3 slot grids (Ted Pella, catalog number: 01816) (Figure 1)
   
   
   Figure 1. 3 slot grids
   
   
5. APEX2-csGBP vector (Addgene, catalog number: 108874) (store at -20 °C)
6. mKate2-P2A-APEX2-csGBP vector (Addgene, catalog number: 108875) (store at -20 °C)
7. APEX2-csChBP vector (Addgene, catalog number: 108876) (store at -20 °C)
8. EGFP-P2A-APEX2-csChBP vector (Addgene, catalog number: 108877) (store at -20 °C)
9. mVenus-N vector with insert of choice cloned into EcoRV/SpeI (Addgene, catalog number: 108887) (store at -20 °C)
10. mVenus-C vector with insert of choice cloned into EcoRV/SpeI (Addgene, catalog number: 108888) (store at -20 °C)
11. Lipofectamine 3000 (Life Technologies, catalog number: L3000015) (store at 4 °C)
12. Bovine hemin (Sigma-Aldrich, catalog number: H9039) (store at 4 °C)
13. 3,3'-diaminobenzidine tetrahydrochloride (DAB) (Sigma-Aldrich, catalog number: D4168) (store at -20 °C)
14. Glutaraldehyde (Electron Microscopy Sciences, catalog number: 16220) (store at 4 °C)
15. Hydrogen peroxide (Sigma-Aldrich, catalog number: H1009) (store at 4 °C)
16. Osmium tetroxide (OsO₄) (ProSciTech, catalog number: C010) (store at 4 °C). Acutely toxic and corrosive.
17. LX112 resin kit (Ladd Research, catalog number: 21310) (store at -20 °C when made up)
18. Sodium cacodylate trihydrate (Sigma-Aldrich, catalog number: C0250) (store at 4 °C)
19. Ethanol (Chem-supply, catalog number: EA043-2.5L-P) 
20. Hydrochloric Acid 32% (RCI, catalog number: RP1104)
21. NaOH
22. KCl
23. Na₂HPO₄
24. KH₂PO₄
25. NaCl
26. Bovine hemin (if necessary) (see Recipes)
27. PBS, pH 7.4 (see Recipes)
28. 2.5% glutaraldehyde in 0.1 M cacodylate buffer (see Recipes)
29. 0.2 M cacodylate buffer (see Recipes)
30. DAB/Cacodylate wash mixture and DAB/cacodylate reaction mixture (see Recipes)
31. Osmium tetroxide solution (see Recipes)
32. Ethanol series for dehydration (see Recipes)
33. LX112 resin ethanol series (see Recipes)
    

Equipment

1. Pliers (JBS, catalog number: 00718811) or similar
2. Oven (SEM, model: 24) or similar
3. PELCO Biowave with PELCO ColdSpot Pro system for temperature control (Ted Pella) (Figure 2)
   
   
   Figure 2. PELCO Biowave
   
   
4. JEM-1011 Transmission Electron Microscope (JEOL) or similar
5. Leica EM UC6 Ultramicrotome (Leica Microsystems) or similar
6. Morada CCD camera (Olympus Soft-Imaging-Solutions) or similar (Figure 3)
   
   
   Figure 3. JEM-1011 electron microscope (left), Leica EM U6 Ultramicrotome (middle), Morada CCD camera (right)
   
   
7. 45° diamond knife (Diatome, supplied by Agar Scientific, catalog number: AGG339-5) (Figure 4)
   
   
   Figure 4. 45° diamond knife
   
   
8. Project specific tissue culture equipment such as a class II biological safety cabinet, CO₂ incubator etc.
9. Fume hood
10. Vortex
    

Software

1. iTEM (EMSIS GmbH) v5.2, build 4768, license required, https://www.emsis.eu/products/software/item
2. Photoshop CC (Adobe), license required, https://www.adobe.com/au/creativecloud.html
   

Procedure