Sean Lauber:Cytokeratin IHC staining

Protocol

1. Cut 3 μM sections, mount on slides, air dry overnight
2. Place slides at 60*C for 45 min
3. Place slides in xylene I for 5 min, then to xylene II for 5 min, then to xylene III for 5 min (rotate xylene and discard xylene I, add fresh to xylene III, after 2 rackings)
4. Transfer to 100% alc I for 10 sec, then to II for 10 sec, then to III for 10 sec
5. Place into freshly prepare methanol solution for 25 min
6. Transfer to 100% alcohol for 10 sec, then to 95% for 10 sec, then to 70% for 10 sec
7. Wash in running tap water for 5 min
8. Rinse in distilled water for 1 min
9. Place slides onto rack and flood with TBS, let sit for 5 min (covered with TBS)
10. Drain & wipe slides, then add enough drops of Proteinase K to cover sample (about 3) for 5 min, cover rack
11. Flood slides 3 times for 5 minutes each with TBS
12. Drain & wipe slides, add enough drops of 5% NGS for 15 min, cover rack
13. Drain & wipe slides, add enough drops of 1:500 dilution of pan-cytokeratin (Dako) for 1 hr
14. Flood slides 3 times for 5 minutes each with TBS
15. Drain & wipe slides, add enough drop of Envision+-rabbit for 30 min (record number of drops)
16. Flood slides 3 times for 5 minutes each with TBS
17. Place in 0.05 M acetate buffer pH 5.0 for 5 min
18. Place in freshly prepared and filtered chromogen/substrate solution for 15 min
19. Place in running tap water for 5 min
20. Rinse in distilled water and counterstain with Mayer's Hxt for 20 sec
21. Wash in running tap water for 1 min
22. Place in TBS for 1 min
23. Mount sections with glycerin

Solutions required

Methanol solution

  200 ml methanol
  10 ml 30% H2O2
  0.5 ml concentrated HCl (add this last)

1:500 pan-cytokeratin

For 2 slides that have 6 pieces of lung (500 μL):

  1 μL of pan-cytokeratin
  500 μL UltraAb diluent

Chromogen/substrate solution

  AEC solution: dissolve 80 mg aminoethylcarbazole in 20.0 ml dimethylformamide in GLASS

  95 ml 0.05 M acetate buffer (pH 5.0)
  5 ml AEC
  mix well and filter
  Just before use add 2 drops 30% H2O2