Materials and Reagents

1. Poly-L-lysine-treated glass slide (Poly-Prep Slides) (Sigma-Aldrich, catalog number: P0425-72EA )
   
2. Coverslips (Matsunami Glass, catalog number: C218181 )
   
3. Dako Pen (Dako, catalog number: S2002 )
   
4. Plastic box (9 cm x 19 cm x 4 cm)
   Note: In this experiment, but any size is OK.
   
5. Kimwipes^® paper (Kimberly Clark)
   
6. Chlamydomonas cells of interest
   
7. Hematocytometer (Erma, catalog number: 03-303-6 )
   
8. Phosphate-buffered Saline (PBS)
   
9. Tween-20 (Santa Cruz, catalog number: sc-29113 )
   
10. PBS-T (PBS with 0.1% Tween-20)
    
11. Paraformaldehyde (Nacalai Tesque, catalog number: 26126-54 )
    
12. Methanol (Nacalai Tesque, catalog number: 21915-64 )
    
13. Globulin-free Bovine Serum Albumin (BSA) (Nacalai Tesque, catalog number: 01281-26 )
    
14. Affinity-purified rabbit primary antibody against LCIB (Yamano et al., 2010)
    
15. Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor^® 488 conjugate (Thermo Fisher, catalog number: A-11001 )
    
16. Mounting medium (Vectorshield, catalog number: H-1300 )
    
17. 4% formaldehyde (see Recipes)
    
18. PBS (see Recipes)
    
19. PBS-T (see Recipes)
    
20. Blocking solution (see Recipes)

Equipment

1. Coplin jar (50 ml size)
   
2. Fluorescence microscopy Axioscope2 (Zeiss) with a specific filter set (excitation bandpass
   480/40 and emission band pass 527/30) or laser scanning confocal microscopy TCS SP8
   (Leica) with a 488 nm laser line
   

Note: these were the instruments used throughout our experiments, but any company is OK.

Procedure