Tunel en células fijadas

TUNEL IN FIXED CELLS

Cannabinoid signaling group

Department of Biochemistry and Molecular Biology I

Faculty of Chemistry
Complutense University of Madrid
28040 Madrid (Spain)
Phone: [phone]
Fax: [phone]

Pág. 1/1

REAGENTS

• 
  PFA (SIGMA):
  
• 
  Cryoprotector: 1,256 g NaH₂PO₄, 4,36 g Na₂HPO₄, solve in 320 ml of PBS; add 240 ml of etilenglycol and 240 ml de glicerol; keep at 4ºC.
  
• 
  BSA (SIGMA)
  
• 
  TritonX100
  
• 
  Buffer TUNEL (Roche 1243276)
  
• 
  rTdT: terminal transferase, recombinat (Roche 03333574001)
  
• 
  dUTP-biotinilated (Roche 1093070)
  
• 
  Streptavidina- Alexa 594 (red) conjugate (Molecular Probes)
  
• 
  Mowiol (Calbiochem 475904)
  
• 
  YOYO-1 (Molecular Probes Y-3601)
  

PROCEDURE

STEP 1. Cells fixation:

4% PFA for 10’

PBS for 5’ (2x)

Add cryoprotector if the procedure will be stopped and freeze to -20º C.

STEP 2. Remove the cryoprotector and wash with PBS for 5‘ (2x).

STEP 3. Permeabilize with PBS + BSA 3mg/ml + 0.25 % TX100 for 15’.

STEP 4. Wash with PBS for 5’ (3x).

STEP 5. Add the mixture of the tunnel reaction,

Stocks

Buffer (5x)

rTdT 75u/ml

dUTP-biotinilated 50nmol (400x)

Complete up to final volume with distilled water.

STEP 6. Incubate 2h to 37º C in wet chamber.

STEP 7. Stop the reaction with 2x SSC for 15’.

STEP 8. Wash with PBS for 5’ (4-5x).

STEP 9. Incubate with Streptavidine Alexa 594 (red) conjugate; 3,5ul of Strep/ml PBS for 2 h in darkness and room temperature.

STEP 10. Wash with PBS for 5’ (4-5x).

STEP 11. Mount with Mowiol + YOYO (green) 1/10000.