Immunohistochemistry Protocol for NB110-97869 (NB110-97869)

1. Dewax and Rehydration: Place the slides in a rack and sequentially pass the rack through following solutions: Histoclear or Xylene twice, 10 minutes each; and then a graded series of 100%, 90%, 70%, and 50% ethanol, 2 minutes each.
2. Wash the slides with tap water for 5 minutes and once with DI water.
3. Antigen Retrieval: treat the slides with 20ug/ml proteinase K in PBS for 15-25 minutes at room temperature.
4. Wash the slides with tap water for 5 minutes and quench the endogenous peroxidase with 3% H2O2 in DI water for 30 minutes
5. Wash the slides with tap water for 5mins and soaking in PBS-Tween20 (0.1%)
6. Block the slides with blocking buffer (25%bovine serum in PBS-Tween20) for at least 10minutes
7. Then incubate the specimen with primary antibody (NB110-97869, dissolved with 200ul PBS) at 1:25-100 dilutions overnight at 4 degree (or room temperature if the signal is too week).
8. Wash the slides with rotation in PBS-Tween20 for 3 times, 5 minutes each.
9. Incubate the specimen with rabbit-anti rat secondary antibody (Vector Laborotary, BA-4001, 0.5mg/ml: 1:50-100) for 30minutes at room temperature.
10. Wash the slides with rotation in PBS-Tween 20 for 3 times, 5 minutes each.
12. Incubate the specimen with Polymer-HRP labeled anti rabbit (Dako: K4010 Envision Kit) for 60 minutes at room temperature.
13. Wash the slides with rotation in PBS-Tween20 for 3 times, 5 minutes each.
14. Perform DAB Color development (reagents available in Dako K4010).
15. Wash with tap water and count stain the nuclear with hematoxylin.

Notes:
1) We recommend that Formalin fixation should be done no longer than overnight at 4 degree.
2) This protocol is for reference only and the final condition should be optimized by the end user.
For Research Use Only