Immunoelectron Microscopy in Chlamydomonas Cells

Materials and Reagents

1. Culture of C. reinhardtii 137mt+ (WT) (we used the strain IPPAS D-298 from the Collection of microalgae of the Institute of Plant Physiology RAS, Moscow)
   
2. Primary antibodies raised in rabbits against the protein of interest (we used antibodies raised against the recombinant Cah-3 protein (α-CA) of C. reinhardtii, Agrisera, Vannas, Sweden)
   
3. Secondary antibodies: Anti-Rabbit IgG conjugated with 10 nm colloidal gold particles (Sigma-Aldrich, catalog number: G7402 )
   
4. Paraformaldehyde (Sigma-Aldrich, catalog number: P6148 )
   
5. LR White embedding kit (Sigma-Aldrich, catalog number: 62662-1EA-F )
   
6. Gelatin capsules (SPI supplies, catalog number: 02308-SS )
   
7. Formvar and carbon-coated nickel grids (SPI supplies, catalog number: 3430N-CF )
   
8. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A7906 )
   
9. Goat serum (Sigma-Aldrich, catalog number: G9023 )
   
10. Rabbit serum (Sigma-Aldrich, catalog number: R4505 )
11. Paraformaldehyde (Sigma-Aldrich, catalog number: P6148 )
    
12. Ethanol
    Optional
    
13. Uranyl acetate (e.g. SPI supplies, catalog number: 02624-AB )
    
14. Lead citrate (e.g. Sigma-Aldrich, catalog number: 15326 )
    
15. NaOH
16. Phosphate buffer saline (PBS) (pH 7.4) (see Recipes)
    
17. Fixation solution (4% paraformaldehyde) (see Recipes)
    
18. Tris-buffer saline (TBS) (pH 7.4) (see Recipes)
    
19. 1% BSA-TBS (see Recipes)
    
20. Uranyl acetate (see Recipes)
    
21. Lead citrate (see Recipes)
    

Equipment

1. 96 well immunology plate (e.g. Greiner Bio-One, catalog number: 650001 )
   
2. Pasteur pipettes with thin tips (ROTHE 306.1)
   
3. Thermostat unit with, at least, 37-55 °C temperature range (e.g. BINDER B28)
   
4. Ultramicrotome (e.g. Reichert, OMU-3, Austria)
   
5. Grid storage box (Sigma-Aldrich, catalog number: G6276 )
   
6. Transmission electron microscope (e.g. Libra-120, Carl Zeiss, Germany)
   
7. Plastic Petri dish or Teflon plate with hydrophobic surface
   
8. Wet chamber
   

Procedure