IMMUNOHISTOCHEMISTRY (NEN BIOTINYL TYRAMIDE AMPLIFICATION)
PERKIN ELMER #NEL700A
Before starting:
 Make 1 L 1X TBS
o 100 ml 10X TBS
o 900 ml dH2O
 TNB
o 0.05 g NEN block reagent
o 10 ml 1x TBS
 heat TBS on stir plate to dissolve blocking reagent; aliquots, store at
-20°C degrees)
 Prepare humid chambers (with Whatman paper and 1X TBS)
 Make 4 L 1XTNT
o 400 ml 10X TBS
o 3600 ml dH2O
o 2 ml Tween-20 (add while stirring!)
Staining process: (use TSA kit, #NEL700A)
1.
Incubate slides at 60°C on slide warmer for at least 1hr.
2.
Deparaffinize and dehydrate (use clean solutions!)
 Xylene, 3x 3 min
 100% EtOH, 3x 2 min
3.
Incubate in 1X TBS, 5 min RT
4.
Perform antigen retrieval, if necessary:
 SODIUM CITRATE
i. 12.5 ml Citrate Buffer pH 6.0 (Invitrogen 00-5000)
ii. 237.5 ml ddH2O
iii. Microwave buffer to about 90 – 95 C (2.5 min)
iv. Place it into preheated waterbath 95 C and wait until temperature is
reached.
v. Put slides in buffer for 10 mins at 95 C
vi. Let solution and slides cool to RT for about 30 mins
5.
Quench in 3% H2O2/ MeOH 10min RT
 25 ml 30% H2O2 + 225 ml MeOH
6.
dH2O, 5min, RT
7.
8.
Circle sections with Pap-pen, rinse in 1X TBS
To block, incubate with 100 l TNB at RT in moist chamber for 30 min.
Updated 4/21/15 by LM
9.
Pour off TNB, and add1 antibody diluted in TNB. Incubate at RT in moist chamber for
1hr
 Diluted 1/50
10. Wash: 1X TNT, 3x 5min at RT (on rocking platform)
11. Add 100 l Biotinylated 2 Ab diluted in TNB. Incubate in moist chamber for 30 min
 Diluted 1/300 for Dako antibodies
12. Wash: 1X TNT, 3x 5min at RT (on rocking platform)
13. Add SA-HRP (from kit) diluted in TNB 1/100. Incubate for 30min RT in moist chamber
14. Wash: 1X TNT, 3x 5min at RT (on rocking platform)
15. Add Biotynil Tyramide (kit), diluted 1/50 in amplification diluent. Incubate 5 min RT in
moist chamber
16. Wash: 1X TNT, 3x 5min at RT (on rocking platform)
17. Add SA-HRP (from kit) diluted in TNB 1/100. Incubate for 30min RT in moist chamber
18. Wash: 1X TNT, 3x 5min at RT (on rocking platform)
19. For HRP detection, use DAB (VECTOR kit)
Vector DAB
To 5 ml dH2O:
 add 2 drops buffer; mix well
 add 4 drops DAB; mix well
 add 2 drops H2O2mix well
 add 2 drops NiCl (optional, if not using hematoxylin counterstain turns DAB
color from brown to black)
20. Incubate until color develops (~2-10min.)
-
Counterstain with Hematoxylin (Invitrogen 00-8001) for 3 min.
-
Wash 5 min in cold tap water
-
Incubate in 1X PBS 1 min
-
Wash in dH2O
-
Dehydrate (1min in EtOH for 3 times, 2 mins in Xylene for 3 times) and coverslip as
usual
Updated 4/21/15 by LM