Immunofluorescence (Especially for Cells Growing on a Coverglass)

Materials and Reagents

1. Primary antibodies: self-produce or commercially order
   Note: For self-produce primary antibodies, optimalize antibody concentration at 1-50 µg/ml); Try 5 µg/ml as a starting point. For commercial primary antibodies, usually 1:1,000 dilution as a starting point, and then adjust the working concentration according to the preliminary data.
   
2. Secondary antibodies (below are the most common secondary antibodies):
   1. Goat anti-rabbit IgG (Life Technologies, Molecular Probes^®/Alexa Fluor^® 488, catalog number: A-11008 )
      
   2. Goat anti-rabbit IgG (Life Technologies, Molecular Probes^®/Alexa Fluor^® 594, catalog number: A-11012 )
      
   3. Goat anti-mosue IgG (Life Technologies, Molecular Probes^®/Alexa Fluor^® 488, catalog number: A-11001 )
      
   4. Goat anti-mosue IgG (Life Technologies, Molecular Probes^®/Alexa Fluor^® 594, catalog number: A-11005 )
      
   5. Goat anti-human IgG (Life Technologies, Molecular Probes^®/Alexa Fluor^® 488, catalog number: A-11013 )
      
   6. Goat anti-human IgG (Life Technologies, Molecular Probes^®/ Alexa Fluor^® 594, catalog number: A-11014 )
      Note: We usually dilute commercial secondary antibodies at 1: 200 as a starting point. Make sure to spin down the stock solution of secondary antibody before dilution to prevent precipitated fluorophores from causing high background. We usually aliquot secondary antibody and stored at -20 °C.
      
3. 4’, 6-diamidine-2-phenylindole dihydrochloride (DAPI) (Boeringer Manheim, 236 276) (make 1 mg/ml stock, 1,000x)
   
4. Paraformaldehyde (16% solution) (Electron Microscopy Sciences, catalog number: 15170 )
   
5. Methanol (Thermo Fisher Scientific, catalog number: BP 1105-4 )
   
6. Coverglasses (12 mm) (Thermo Fisher Scientific, catalog number: 12-545-82 )
   
7. Poly-L-lysine (MW 150,000-300,000) (Sigma-Aldrich, catalog number: P1399 )
   
8. Fluormount-G^TM (mounting solution) (SouthernBiotech, catalog number: 0100-01 )
   
9. Nitric acid
   
10. Hydrochloric acid
    
11. Liquid nitrogen
    
12. PBS-BT solution (see Recipes)
    
13. Normal goat serum (The Jackson Laboratory, catalog number: 005-000-001 ) (see Recipes)
    

Equipment

1. Glass beaker
   
2. Petri dishes
   
3. UV-irradiate
   
4. 24-well plate
   
5. 6-well plate
   
6. 60 mm dish
   
7. Parafilm
   

Procedure