3 diaminobenzidine dab chromogen

An immunohistochemical staining of formalin-fixed, paraffin-embedded mouse tissues was performed using rabbit monoclonal Ki-67 antibody (Abcam, Cambridge, MA, Cat# ab16667, Lot# GR3185488–3, 1 mg/ml)) at a 1:150 dilution. Further, the sections were incubated with
Rabbit-on-Rodent HRP-Polymer (BioCare Medical, Concord, CA) for 30 minutes at room temperature. The antigen-antibody complex was visualized using 3-diaminobenzidine (DAB) chromogen (Dako North America, Inc., Carpenteria, CA) for 6 minutes at room temperature. Finally, the sections were counterstained with hematoxylin, dehydrated through graded ethanol, cleared in xylene, and coverslipped.

Antibodies used for IF and IHC are listed in supplementary material, Table S1. Paraffin sections cut at 5 μm were mounted on Fisherbrand Superfrost slides (Pittsburgh, PA, USA), incubated for 60 min at 60 °C and then deparaffinized and rehydrated. Epitope retrieval was performed in a Decloaking Chamber (Biocare Medical, Pacheco, CA, USA) under pressure for 5 min using citrate buffer pH 6.0, followed by a 10-min cooling down period. Endogenous peroxidase was blocked using 3% H₂O₂ for 10 min, followed by incubation with primary antibody for 40 min followed by Mach 2 HRP Polymer BioCare (Concord, CA, USA) for 30 min, and 3-diaminobenzidine (DAB)+ chromogen (Dako, Carpinteria, CA, USA) for 5 min. Immunohistochemical staining was performed using an IntelliPATH FLX Automated Stainer (Biocare Medical, Pacheco, CA, USA) at room temperature (RT). Light hematoxylin counterstaining was performed, after which the slides were dehydrated, cleared, and mounted using a permanent mounting medium.