Staining procedure with paraffin sections with antibodies to laminin, fibronectin,
collagen type IV
Solutions:
Trypsin(Sigma, Cat# T7168)
1X Automation Buffer (Biomeda Corp., cat#M30); dilute 10X concentrate with dH2O
Vector VIP peroxidase substrate kit (Vector, Cat# SK-4600)
Biotinylated rabbit anti-rat IgG, mouse serum absorbed (Vector, Cat# BA-4001)
Biotinylated goat anti-rabbit IgG (Vector, Cat# BA-1000)
Vectastain Elite ABC-peroxidase kit (Vector, Cat# PK-6100)
Blocking buffer: 1x PBS, 5% BSA, 2% rabbit or goat serum
Procedure:
1. Bake slides in 56°C oven for one hour.
2. Wash in xylene for 10 minutes.
3. Wash in 100% EtOH for 10 minutes.
4. Wash in 70% EtOH for 10 minutes.
5. Air dry and outline sections with PAP PEN (Jersey Lab & Glove, Cat# Pen513).
6. Rehydrate in PBS for 5 minutes.
7. Treat with 0.1% trypsin in PBS for 30 minutes at 37°C.
8. Wash under running tap water for 10 minutes.
9. Wash in 70% EtOH for 5 minutes.
10. Wash in 0.3% H2O2 in methanol for 30 minutes.
11. Wash in 70% ethanol for 5 minutes.
12. Wash in 1X automation buffer for 5 minutes.
13. Incubate with blocking buffer for 30 minutes.
14. Blot excess serum from sections.
15. Incubate with rabbit anti-laminin IgG (1:50 dilution), fibronectin (1:50 dilution), or collagen type IV (1:50 dilution) for two hours at room temperature in the humidified box. All the antibody stocks are 1 mg/ml concentration.
16. Wash in 1X automation buffer for 5 minutes.
17. Incubate with biotinylated goat anti-rabbit IgG (1:100 dilution of the 1.0 mg/ml stock) for 30 minutes.
18. Wash twice in 1X automation buffer for 5 minutes, each time.
19. Incubate with ABC reagent for 30 minutes (prepare 30 min. before use).
20. Wash in 1X automation buffer for 5 minutes.
21. Incubate with peroxidase substrate solution (Vector VIP Kit) for 2-15 minutes.
22. Wash in tap water for 5 minutes.
23. Dehydrate through a series of EtOH baths (50% EtOH for 15 seconds, 70% EtOH for 15 seconds, 95% EtOH 2X 15 seconds each, 100% EtOH for 15 seconds and for 30 seconds), and finally clear in xylene 3X 5 minutes each.
24. Mount with permount.
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