Difference between revisions of "TSA Staining on axolotl-tissue"
Jump to navigation
Jump to search
| Line 1: | Line 1: | ||
== Material == | == Material == | ||
| − | * TNT wash buffer: 0.1M TRIS-HCL, pH 7.5 | + | * TNT wash buffer: |
| − | + | 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 | 0.15M NaCl | ||
20% goat serum | 20% goat serum | ||
| − | + | ** for diluting the AB: | |
| − | ** for diluting the AB: 0.1M TRIS-HCL, pH 7.5 | + | 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 | ||
| − | |||
== Procedure == | == Procedure == | ||
Revision as of 08:28, 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