IMMUNOFLUORESCENCE ON CELLS IN CULTURE (2)

(All incubations at 22°C on rotating-plate for good mixing)

• Cells cultured on coverslips 12 mm (100.000 cells/well)
   
• Wash 3 x 5 min with PBS-Ca²⁺, 37°C 

(if necessary permeabilisation before fixation : 0.1 % Triton X-100 in PBS for 5 min, Wash 2 x 5 min PBS-Ca²⁺)

• Fixation in 4% formaldehyde, 0.1 % glutaraldehyde in 0.1M phosphate buffer at 22°C, 20 min

(if necessary permeabilisation after fixation : 0.5 % Triton X-100 in PBS for 20 min, Wash 2 x 5 min PBS-Ca²⁺)
 

• Quenching at 22°C:
  • 0.1% glycin in PBS-Ca²⁺, 15 min
  • 1mg/ml NaBH₄ in PBS-Ca²⁺, 15 min (+ rinse 4 times with PBS-Ca²⁺)
  • 0.1% albumin in PBS, 15 min
     
• Incubation: first antibody 60 min in PBS-Ca²⁺ with 0.1% albumin
   
• Wash 5 x 5 min in PBS-Ca²⁺ with 0.1% albumin
   
• Incubation: second antibody 60 min in PBS-Ca²⁺ with 0.1% albumin in dark
   
• Wash 5 x 5 min in PBS-Ca²⁺ with 0.1% albumin
   
• Mounting coverslip with Mowiol/DABCO