- Proteins and Peptides
- Lysates and Cell Lines
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.
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.