Immunohistochemistry: Sca1 (by Masanobu Ohishi)
Before starting:
· Make 1 L 1X TBS
o 100 ml 10X TBS
o 900 ml dH2O
· 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, Perkin Elmer #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) Rehydrate in 1X TBS, 5 min RT
- At this time, prepare PK
4) Circle sections with Pap-pen
5) Proteinase K 20ug/ml, 15min RT in moist chamber
- 6 ml of 10mg/ml in 3 ml dH2O
6) 3% H2O2/ MeOH 10min RT
- 25 ml 30% H2O2 + 225 ml MeOH
7) dH2O, 5min, RT
8) Equilibrate sections by rinsing with 1X TBS
9) Incubate with TNB at RT in moist chamber for 30 min.
10) 1° antibody diluted in TNB, RT in moist chamber for 1hr
- Sca1 (BD 557403) diluted 1/500 in TNB
11) Wash: 1X TNT, 3x 5min at RT (on rocking platform)
12) Biotinylated 2° Ab diluted in TNB, in moist chamber for 30 min
- antiRat IgG,1/300 (Dako, #E0468)
13) Wash: 1X TNT, 3x 5min at RT (on rocking platform)
14) SA-HRP (from kit) diluted in TNB 1/100 30min RT in moist chamber
15) Wash: 1X TNT, 3x 5min at RT (on rocking platform)
16) Biotynil Tyramide (kit), diluted in amplification diluent, 1:50); 5 min RT in moist chamber
17) Wash: 1X TNT, 3x 5min at RT (on rocking platform)
18) SA-HRP (from kit) diluted in TNB 1/100 30min RT in moist chamber
19) Wash: 1X TNT, 3x 5min at RT (on rocking platform)
20) 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)
21) Incubate until color develops (2-10min.)
22) Wash 5 min in dH2O.
23) Counterstain with Hematoxylin (Invitrogen 00-8001) for 3 min.
24) Wash 5 min in cold tap water
25) Incubate in 1X PBS 1 min
26) Wash in dH2O
27) Dehydrate (1min in EtOH for 3 times, 2 mins in Xylene for 3 times) and coverslip as usual
Updated 4/21/15 by LM