Immunofluorescence staining of LNCaP cells  - Elizabeth Rico-Bautista, April 2006

 

  1. Rinse coverslips with sterile water
  2. Wash with EtOH
  3. Wash with sterile water
  4. Dip the slips into the poly-lysine solution (0.01%). Incubate 15 min
  5. Wash with sterile water (3 times)
  6. Allow to air dry
  7. Expose to UV light for 45 min

 

Immunofluorescence staining

 

  1. Grow the cells in sterile coverslips overnight (LNCaP cells 40000-50000 cells / coverslip)
  2. Treatment of the cells (different drugs or transfection)
  3. Take the coverslip and dip it into PBS (be careful not to detach cells)
  4. Fixation: Use para-formaldehyde 4% in PBS (Fresh). Take the coverslip and dip it into the fixation solution. Incubation 30 min at RT. (you can use a 12-well tissue culture plate)
  5. Permeabilization: Remove the fixation solution by aspiration (gently) and permeabilize the cells by adding PBS containing 0.1% Triton X100. Incubate for five minutes at RT (Repeat). Do not allow the coverslips to dry.
  6. Remove the detergent solution and add the blocking solution (milk 5% in PBS, 0.1% Triton). Incubate for 1h at RT
  7. Take the coverslips from the 12-well dish and place them on a piece of parafilm in a 15 cm tissue culture dish (dish wrapped with aluminum foil). Do not allow the coverslips to dry.
  8. Remove the excess of blocking solution and add the first antibody (50 μl/ coverslip). (Use blocking solution for the dilution of the first antibody). Incubate 1h at 37°C. (Optional: place a wet filter paper on the lid of the incubation dish to prevent drying).
  9. Remove the first antibody and wash with blocking solution 3-5 times (700 μl/coverslip)
  10. Add the secondary antibody (50 μl/ coverslip). (Use blocking solution for the dilution of the secondary antibody). Incubate 50 min to 1h at 37°C.
  11. Remove the secondary antibody and wash the coverslips in five changes of PBS-triton (5 x 3 min)
  12. Add 50 μl of Hoechst solution, 1μg/ml in PBS (Stock 1mg/ml) and incubate 2 min.
  13. Wash twice with PBS
  14. On a clean microscope slide place a drop (3.5 μl) of Fluoromount (mounting media). Take the coverslip using fine-tipped forceps and drain the last wash by touching the edge of the coverslip to a clean paper towel. Invert the coverslip and place on the drop of mounting medium with the cell side down. Gently and carefully allow the coverslip to fall on the drop. Seal the coverslip using nail polish.
  15. When the nail polish is dried, wash the top of the coverslip with water. Store slide in the dark.
  16. Observe and take photographs under the fluorescence microscope.

 

Secondary antibodies:

Alexa Fluor 594 goat anti-mouse IgG: (Ex: 590 nm; Em: 617 nm). Working dilution 1:200

Alexa Fluor 488 donkey anti-rabbit IgG: (Ex: 495 nm; Em: 519 nm). Working dilution 1:200