Richards: Cytokeratin IHC staining
Protocol
Deparaffinize
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
Stain
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
