| TUNEL PROCEDURE IN BOVINE EMBRYOS
Fabiola Paula-Lopes, F. Dean Jousan, Zvi Roth, P. Soto, and P. J. Hansen
Dept. of Animal Sciences, University of Florida and Lehrstuhl für Molekulare Tierzucht und Biotechnologie, Ludwig-Maximilians-Universität Müenchen (FPL)
http://www.animal.ufl.edu/hansen/protocols/tunel.htm
Materials
8% (w/v) paraformaldehyde stock solution: Dissolve 8 g of powdered paraformaldehyde in 100 ml water. Heat and stir (55-60 C – do not go higher). Add a few drops of 2 N sodium hydroxide until the solution clears. Make fresh each day. Alternatively, 8% paraformaldehyde can be purchased from Electron Microscopy Sciences as a custom formulation in 4 ml aliquots (cat. no. 15710-SP). Throw away whatever is not used in one day.
4% (v/v) paraformaldehyde: 1:1 solution of 8% paraformaldehyde stock solution and 0.2 M PBS. Make up on the day of use.
Microscope slides: dip the slides in 1:10 poly-l-lysine solution (Sigma P8920) for 2 minutes. Allow the slides to dry.
DNase (50 U/ml) - The stock is RQ1-RNA free DNase, Promega cat. # 610A, concentration 1U/µl. Dilute 10 µl DNase with 190 µl PBS/PVP0µl. Use fresh.
RNase (50µg/ml) - Use RNase A (heat treated), QIAGEN, Germany, cat.#1901, concentration 100mg/ml as a stock. Dilute 1 µl RNase A (stock) with 999 µl PBS/PVP and then dilute this solution 1:1 with PBS/PVP to obtain a working solution (Use fresh).
Mounting medium and antifade: ProLong Antifade Kit (Molecular Probes P-7481)
TUNEL reaction mixture (In Situ Cell Death Detection Kit, Fluorescein: Boehringer Mannheim; Cat. No. 1684795): Remove 100 µl Label Solution from bottle 2 for two negative controls. Add total volume of bottle 1 (50 µl) to the remaining 450 µl Label Solution in bottle 2 to obtain 500 µl TUNEL reaction mixture. Mix well to equilibrate components.
上一篇：TUNEL Assay Protocol [Rosen Lab, Baylor College of Medicine] 下一篇：In Situ Apoptosis Detection by TUNEL labeling (Frozen Section) [Wilcox Lab ,Emor
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