View source for 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