Diff of 11.1.3 TUNEL staining

The added line is THIS COLOR.
The deleted line is THIS COLOR.
Go to 11.1.3 TUNEL staining.
[[FrontPage]]

*11.1.3 TUNEL staining

The terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method labels fragmented DNA which is a characteristic of the apoptotic cells.

Yuki Nakatani~
Tokyo Institute of Technology

**11.1.3.1 Reagents

-In Situ Cell Detection Kit (AP) by Roche
-4% paraformaldehyde
-methanol 
-PBST: PBS + 0.1% Triton X-100
-10&micro;g/ml proteinase K in PBS
-0.1% sodium citrate, 0.1% Triton X-100 (freshly prepared)
-Blocking solution: 3% BSA, 5% lamb serum in 0.1M Tris-HCl (pH7.5)
-AP buffer: 100mM Tris-HCl (pH9.5), 100mM NaCl, 50mM MgCl2, 0.1% Triton X-100, 1mM Levamisole
-AP staining solution: 100mM Tris-HCl (pH9.5), 100mM NaCl, 50mM MgCl2, 1mM Levamisole, 337.5&micro;g/ml NBT, 175&micro;g/ml BCIP

**11.1.3.2 Method

+Fins are fixed with 4% paraformaldehyde at 4&ordm;C, O/N
+Wash with PBST
+Dehydrate gradually through a methanol series (25-50-75-100% methanol in PBST)
+Store in 100% methanol at -20&ordm;C
+Wash with PBST
+10 &micro;g/ml proteonase K treatment at RT for 5 min
+PBST wash
+Refix in 4% paraformaldehyde at RT for 20 min
+Wash with PBST
+0.1% sodium citrate, 0.1% Triton X-100 on ice for 15 min
+Wash with PBST
+Replace PBST with  reaction mixture (solution 1: solution 2 (kit) = 1:9)
+Incubate at 37&ordm;C for 1 hr
+Wash with PBST
+Incubate in blocking solution at RT for 1 hr
+Incubate in 1:2000 AP-conjugated anti-fluorescein antibody at 4&ordm;C, O/N
+Wash with PBST
+Wash with AP buffer
+Incubate in AP staining solution until signals are detected
+Stop reaction with PBST when signals are detected

**11.1.3.3. Reference

Barrallo-Gimeno A, Holzschuh J, Driever W, Knapik EW. (2004). Neural crest survival and differentiation in zebrafish depends on mont blanc/tfap2a gene function. Development 131 (7), 1463-1477.