TSA Staining on axolotl-tissue: Difference between revisions
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== Material == | == Material == | ||
* TNT wash buffer: | * TNT wash buffer: 0.1M TRIS-HCL, pH 7.5; 0.15M NaCl; 0.05% Tween20 | ||
0.1M TRIS-HCL, pH 7.5 | |||
0.15M NaCl | |||
0.05% Tween20 | |||
* TNB buffer: | * TNB buffer: | ||
** for blocking: | ** for blocking: 0.1M TRIS-HCL, pH 7.5; 0.15M NaCl; 20% goat serum | ||
0.1M TRIS-HCL, pH 7.5 | ** for diluting the AB: 0.1M TRIS-HCL, pH 7.5; 0.15M NaCl; 10% goat serum | ||
0.15M NaCl | |||
20% goat serum | |||
** for diluting the AB: | |||
0.1M TRIS-HCL, pH 7.5 | |||
0.15M NaCl | |||
10% goat serum | |||
* quenching solution (QS): 3% H2O2 in PBS | * quenching solution (QS): 3% H2O2 in PBS | ||
Latest revision as of 08:49, 1 June 2015
Material
- TNT wash buffer: 0.1M TRIS-HCL, pH 7.5; 0.15M NaCl; 0.05% Tween20
- TNB buffer:
- for blocking: 0.1M TRIS-HCL, pH 7.5; 0.15M NaCl; 20% goat serum
- 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
- wash once for 5 min @ RT with TNT
- quench for 15 min @ RT with QS
- wash once again for 5 min @ RT with TNT
- block for 30 min in TNB-20% GS@ RT
- primary AB:
- supernatant of HC: use pure
- purified AB: dilute 1:500in TNB-10% GS
- centrifuge for 10 min @ 13k
- incubate the primary AB o/n at 4ºC
- wash slides 3 times for 5 min @ RT
- dilute the HRP-AB 1:1000 in TNB-10% GS and centrifuge for 10 min @ 13k
- incubate the HRP-AB for 2 hours @ RT
- wash slides 3 times for 5 min @ RT with TNT
- dilute the Thyramide stock 1:50 with Amplification diluent
- incubate 150 µl per slide of the Thyramide dilution for 3- 10 min (best: 6 min)
- wash 3 times for 5 min @ RT with TNT
- add Hoechst 1:1000 in TBS/ Tween for 10 min @ RT
- wash once for 5 min @ RT
- mount