TSA Staining on axolotl-tissue

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

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:
   1. supernatant of HC: use pure
   2. purified AB: dilute 1:500in TNB-10% GS
6. centrifuge for 10 min @ 13k
7. incubate the primary AB o/n at 4ºC
8. wash slides 3 times for 5 min @ RT
9. dilute the HRP-AB 1:1000 in TNB-10% GS and centrifuge for 10 min @ 13k
10. incubate the HRP-AB for 2 hours @ RT
11. wash slides 3 times for 5 min @ RT with TNT
12. dilute the Thyramide stock 1:50 with Amplification diluent
13. incubate 150 µl per slide of the Thyramide dilution for 3- 10 min (best: 6 min)
14. wash 3 times for 5 min @ RT with TNT
15. add Hoechst 1:1000 in TBS/ Tween for 10 min @ RT
16. wash once for 5 min @ RT
17. mount