Immunohistochemical Staining of Paraffin Sections

Paraffin-embedded tissues were cut into 4-5 μm sections using a microtome. After de-paraffinization and hydration, antigen retrieval was conducted by boiling the slides in citrate buffer for 90 s, followed by the incubation with 3% hydrogen peroxide for 10 min. After 3 washes in TBST buffer, the sections were blocked for 1 h in TBST with 5% normal goat serum, and then probed with primary antibodies (1:500) overnight at 4 °C. Following washing with TBST buffer, the sections was soaked with 1–3 drops of SignalStain® Boost Detection Reagent (Cell Signaling Technologies, Inc.) and incubated in a humidified chamber for 30 min at room temperature. The signal was developed using 400 μl SignalStain® substrate (Cell Signaling Technologies, Inc.) for 5 min. The sections were mounted with coverslip using mounting medium (Cell Signaling Technologies, Inc.) before being imaged under a bright-field microscope [23 (link), 24 (link)].