Immunohistochemistry-Paraffin Protocol for CD8 alpha (NBP2-12183)

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 sections with proteinase K (use manufacturers recommendation) 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 10 minutes
7) Then incubate the specimen with primary antibody (NBP2-12183, dissolved with 200ul PBS) at 1:25-100 dilutions overnight at 4 degrees C (or room temperature if the signal is too weak).
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 for 30 minutes at room temperature.
10) Wash the slides with rotation in PBS-Tween 20 for 3 times, 5 minutes each.
11) Incubate the specimen with Polymer-HRP labeled anti rabbit (Dako: K4010 Envision Kit) for 30 minutes at room temperature.
12) Wash the slides with rotation in PBS-Tween20 for 3 times, 5 minutes each.
13) Perform DAB color development (reagents available in Dako K4010).
14) Wash with tap water and counterstain the nuclei with hematoxylin.

Notes:
1. If you use buffered formalin fixatives, we recommend that fixation should be done no longer than overnight (4 degrees C preferred).
2. This protocol is for reference only and the final conditions should be optimized by the end user.