Difference between revisions of "TSA Staining on axolotl-tissue"
| Line 1: | Line 1: | ||
== Material == | == Material == | ||
| − | + | * TNT wash buffer: 0.1M TRIS-HCL, pH 7.5 | |
0.15M NaCl | 0.15M NaCl | ||
0.05% Tween20 | 0.05% Tween20 | ||
| − | + | * TNB buffer: | |
a) for blocking: 0.1M TRIS-HCL, pH 7.5 | a) for blocking: 0.1M TRIS-HCL, pH 7.5 | ||
0.15M NaCl | 0.15M NaCl | ||
| Line 13: | Line 13: | ||
10% goat serum | 10% goat serum | ||
| − | + | * quenching solution (QS): 3% H2O2 in PBS | |
Revision as of 08:12, 1 June 2015
Material
- TNT wash buffer: 0.1M TRIS-HCL, pH 7.5
0.15M NaCl 0.05% Tween20
- TNB buffer:
a) for blocking: 0.1M TRIS-HCL, pH 7.5 0.15M NaCl 20% goat serum
b) for diluting the AB: 0.1M TRIS-HCL, pH 7.5 0.15M NaCl 10% goat serum
- quenching solution (QS): 3% H2O2 in PBS
Procedure
1) wash once for 5 min @ RT with TNT 2) quench for 15 min @ RT with QS 3) wash once again for 5 min @ RT with TNT 4) block for 30 min in TNB-20% GS@ RT 5) primary AB: a) supernatant of HC: use pure b) purified AB: dilute 1:500in TNB-10% GS
- centrifuge for 10 min @ 13k 6) incubate the primary AB o/n at 4ºC 7) wash slides 3 times for 5 min @ RT 8) dilute the HRP-AB 1:1000 in TNB-10% GS and centrifuge for 10 min @ 13k 9) incubate the HRP-AB for 2 hours @ RT 10) wash slides 3 times for 5 min @ RT with TNT 11) dilute the Thyramide stock 1:50 with Amplification diluent 12) incubate 150 µl per slide of the Thyramide dilution for 3- 10 min (best: 6 min) 13) wash 3 times for 5 min @ RT with TNT 14) add Hoechst 1:1000 in TBS/ Tween for 10 min @ RT 15) wash once for 5 min @ RT 16) mount