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
- 12.5 ml Citrate Buffer pH 6.0 (Invitrogen 00-5000)
- 237.5 ml ddH2O
- Microwave buffer to about 90 – 95° C (2.5 min)
- Place it into preheated waterbath 95° C and wait until temperature is reached.
- Put slides in buffer for 10 mins at 95° C
- 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. Circle sections with Pap-pen, rinse in 1X TBS
8. To block, incubate with 100 ml TNB at RT in moist chamber for 30 min.
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 ml 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