Cannabinoid signaling group
Department of Biochemistry and Molecular Biology I
Faculty of Chemistry
Complutense University of Madrid
28040 Madrid (Spain)
Phone: +34 913944668
Fax: +34 913944672
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REAGENTS
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PFA (SIGMA):
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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.
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BSA (SIGMA)
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TritonX100
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Buffer TUNEL (Roche 1243276)
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rTdT: terminal transferase, recombinat (Roche 03333574001)
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dUTP-biotinilated (Roche 1093070)
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Streptavidina- Alexa 594 (red) conjugate (Molecular Probes)
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Mowiol (Calbiochem 475904)
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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.
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