TSA Staining on axolotl-tissue: Difference between revisions

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Solutions:
== 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
0.15M NaCl
* TNB buffer:
0.05% Tween20
** 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


- TNB buffer:
== Procedure ==
a) for blocking: 0.1M TRIS-HCL, pH 7.5
# wash once for 5 min @ RT with TNT
0.15M NaCl
# quench for 15 min @ RT with QS
20% goat serum
# wash once again for 5 min @ RT with TNT
 
# block for 30 min in TNB-20% GS@ RT
b) for diluting the AB: 0.1M TRIS-HCL, pH 7.5
# primary AB:  
0.15M NaCl
## supernatant of HC: use pure
10% goat serum
## purified AB: dilute 1:500in TNB-10% GS
 
# centrifuge for 10 min @ 13k
- quenching solution (QS): 3% H2O2 in PBS
# 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
Procedure:
# incubate the HRP-AB for 2 hours @ RT
 
# wash slides 3 times for 5 min @ RT with TNT
1) wash once for 5 min @ RT with TNT
# dilute the Thyramide stock 1:50 with Amplification diluent
2) quench for 15 min @ RT with QS
# incubate 150 µl per slide of the Thyramide dilution for 3- 10 min (best: 6 min)
3) wash once again for 5 min @ RT with TNT
# wash 3 times for 5 min @ RT with TNT
4) block for 30 min in TNB-20% GS@ RT
# add Hoechst 1:1000 in TBS/ Tween for 10 min @ RT
5) primary AB: a) supernatant of HC: use pure
# wash once for 5 min @ RT
b) purified AB: dilute 1:500in TNB-10% GS
# mount
 
- 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

Latest revision as of 08:49, 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

  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
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