Difference between revisions of "TSA Staining on axolotl-tissue"

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# 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: a) supernatant of HC: use pure
+
# primary AB:
−
b) purified AB: dilute 1:500in TNB-10% GS
+
## 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
−
6) incubate the primary AB o/n at 4ºC
+
# incubate the primary AB o/n at 4ºC
−
7) wash slides 3 times for 5 min @ RT
+
# 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
+
# 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
+
# incubate the HRP-AB for 2 hours @ RT
−
10) wash slides 3 times for 5 min @ RT with TNT
+
# wash slides 3 times for 5 min @ RT with TNT
−
11) dilute the Thyramide stock 1:50 with Amplification diluent
+
# 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)
+
# 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
+
# wash 3 times for 5 min @ RT with TNT
−
14) add Hoechst 1:1000 in TBS/ Tween for 10 min @ RT
+
# add Hoechst 1:1000 in TBS/ Tween for 10 min @ RT
−
15) wash once for 5 min @ RT
+
# wash once for 5 min @ RT
−
16) mount
+
# mount

Revision as of 08:17, 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

  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. a) supernatant of HC: use pure
    2. b) 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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