TSA Staining on axolotl-tissue

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

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