CD34 Antibody – Rabbit Monoclonal

IHC-P dilution 1:100

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 citrate buffer, pH 6.0* and incubate at 95-97 degree C in water bath for 25 minutes.
7. Remove the staining to room temperature and let the slide to cool (in citrate buffer, pH 6.0) 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 for 1 hour in the closed wet chamber.
READY TO USE (RTU):
Incubate the section with primary antibody (ready to use) for 1 hour in a 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). Micropolymer-HRP detection kit rabbit/mouse dual is suggested
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

Citrate Buffer (10mM Citric Acid, 0.05% Tween-20, pH 6.0):
Citric acid (anhydrous) ---------- 1.92 g; Distilled water ----------- 1000 ml Mix to dissolve in 700 ml of distilled water. Adjust pH to 6.0 with 1M NaOH 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 4 degree C for longer storage.