Sean Lauber:IHC development
The following is a generic procedure to determine an appropriate IHC staining protocol for fibronectin. I tried two different antigen retrieval methods here (steaming and enzymatic).
1. Cut 3 μM sections, mount on slides, air dry overnight
2. Place slides at 60*C for 40 min
- Remove the following reagents to warm to RT: UltraAb diluent, H2O2, 5% NGS, Proteinase K, Envision, Acetate buffer
3. Place slides in xylene I for 5 min, then to xylene II for 5 min, then to xylene III for 5 min
- Prepare the methanol solution, label it
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
- Prepare steamer to warm up for 10 min (set it to 55). Fill steamer to knob with tap water.
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)
- Allow one set of slides to sit in the TBS while the other set steamed. Heat citrate buffer (mark) in microwave for 2 min
- Steamer: put slides into hot citrate buffer and steam for 45 min, then let cool for 5 min (at this point do the next step)
10. Drain & wipe slides, then add enough drops of Proteinase K to cover samples (about 3 drops - record drops) for 5 min
11. Flood slides 3 times for 5 minutes each with TBS
12. Drain & wipe slides, add enough drops of 5% NGS for 20 min, cover rack
13. Drain & wipe, add enough drops of 0 (d. negative) , 1:100, 1:250, 1:625 dilution of fibronectin (Abcam, ab2413) for 1 hr
- Add 10 ul to 1 ml UltraAb (1:100), 4 ul to 1 ml UltraAb (1:250), and 1.6 ul to 1 ml UltraAb (1:625)
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 45 sec
21. Wash in running tap water for 1 min
22. Place in TBS for 1 min
23. Mount sections with glycerin
