Difference between revisions of "TSA Staining on axolotl-tissue"
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| Line 21: | Line 21: | ||
# wash once again for 5 min @ RT with TNT | # wash once again for 5 min @ RT with TNT | ||
# block for 30 min in TNB-20% GS@ RT | # block for 30 min in TNB-20% GS@ RT | ||
| − | # primary AB: | + | # primary AB: a) supernatant of HC: use pure |
| − | + | b) purified AB: dilute 1:500in TNB-10% GS | |
| − | |||
# centrifuge for 10 min @ 13k | # centrifuge for 10 min @ 13k | ||
# incubate the primary AB o/n at 4ºC | # incubate the primary AB o/n at 4ºC | ||
Revision as of 08:22, 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
- 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: a) supernatant of HC: use pure
b) 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