Sean Lauber:Fibronectin IHC staining: Difference between revisions
(New page: 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, ...) |
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Latest revision as of 06:19, 8 July 2013
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
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 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) and 1:200 dilution of fibronectin (Abcam, ab2413) 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 45 sec
21. Wash in running tap water for 1 min
22. Place in TBS for 1 min
23. Mount sections with glycerin
