Cytokeratin 5/6 (KRT5) Antibody – Rabbit Monoclonal

Dilution: 1:100 - 1:200

IHC-P Protocol - Instruction Manual:
1. Deparaffinize the section in 3 changes of xylene, 10 minutes each.
2. Wash the section in 96%, 80% and 70% ethyl alcohol for 10 minutes each.
3. Rinse in distilled water, 2 x 5 minutes.
4. Block the endogenous peroxidase by incubating the tissue in 3% hydrogen peroxide (H₂O₂) for 10 minutes.
5. Wash in distilled water, 2 x 5 minutes.
6. For antigen retrieval: Immerse the slide in Tris-EDTA buffer, pH 9.0 and incubate at 95-97ºC in water bath for 25 minutes.
7. Remove the staining to room temperature and let the slide to cool (in Tris-EDTA buffer, pH 9.0) for 15 minutes.
8. Rinse in distilled water, 2 x 5 minutes.
9. Wash in PBS (phosphate buffer saline, pH 7.0-7.5) supplemented with 0.05% of Tween-20 (buffer A), 2 x 5 minutes.
10. CONCENTRATED: Incubate the section with primary antibody at the dilution 1:100 - 1:200 for 1 hour in the closed wet chamber.
11. Wash 3 x 5 minutes with buffer A.
12. Apply the secondary antibody (the protocol depends on the supplier), and proceed to standard immunohistochemistry protocol (HRP - Peroxide - DAB).
13. Wash 3 x 5 minutes with buffer A.
14. Apply the chromogen (DAB), 1 - 3 minutes.
15. Wash in water, 2 x 5 minutes.
16. Stain in hematoxylin for 5 minutes.
17. Wash in distilled water, 3 x 2 minutes.
18. Mount the slide for observation.

* Tris-EDTA Buffer (10mM Tris Base, 1mM EDTA solution, 0.05% Tween-20, pH 9.0):
Tris ----------- 1.21 g; EDTA ---------- 0.37 g; Distilled water ------------ 1000 ml
Mix to dissolve in 700 ml of distilled water. Adjust pH to 9.0 with 1M HCl and then add 0.5 ml of Tween-20 and mix well. Adjust the final volume to 1 liter with distilled water. Store this solution at room temperature for 3 months or at +4ºC for longer storage.

Ventana Protocol - Instruction Manual:
(Short Application Protocol for Ventana Benchmark Slide Staining System)
1. Deparafinization (Enter).
2. Heating glass (72°C) with the medium temperatures. Deparafinization.
3. Prolonged deparafinization (Enter).
4. Cell conditioning (Enter).
5. ULTRA Conditioner #1 (Enter).
6. Heating glass (96°C), incubation 8 min (Cell conditioner #1).
7. ULTRA CC1 solution application – 20 min (Enter).
8. Titration (Enter).
9. Hand apply - primary antibody. Incubation 32 min.
10. UltraWash (Enter).
11. Nuclear stain (Enter).
12. Hematoxylin application - one drop (nuclear stain), cover and incubate 8 min.
13. After nuclear stain (Enter).
14. Bluing reagent application, one drop. After nuclear stain, cover and incubate 4 min.