TSA Staining on axolotl-tissue

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