Signalstain boost detection reagent hrp mouse

To examine protein localization and expression, the formalin-fixed tissues were de-paraffinized. After deparaffinization and dehydration of paraffin sections, heat-induced epitope retrieval was performed using a pressure cooker and sodium citrate buffer (10 mM sodium citrate, 0.05% tween-20, pH 6.0). Once boiled, slides were transferred from PBS to the sodium citrate buffer in pressure cooker for 10 min. Slides were then cooled to room temperature for 30 min, and permeabilized with permeabilization buffer containing 0.3% triton-100 in PBS for 10 min. To block endogenous peroxidase activity, slides were incubated in 3% hydrogen peroxide for 10 min and blocked with animal-free blocking solution (Cat# 15019; Cell Signaling, Inc.). After blocking, slides were incubated overnight at 4°C with mouse monoclonal anti-TGF-β₁ (1:50; Cat# sc-130348; Santa Cruz, Inc.) or rabbit polyclonal anti-TGF-β₁ receptor (1:100; Cat# ab235178; Abcam, Inc.) and subsequently incubated with SignalStain Boost Detection Reagent (HRP mouse; Cat# 8125; or HRP Rabbit; Cat# 8114; Cell Signaling, Inc.) for 30 min at room temperature. Slides were then incubated for 2–10 min with SignalStain DAB (Cat# 8059; Cell Signaling, Inc.), immersed in distilled H₂O, stained with hematoxylin (Cat# 14166; Cell Signaling, Inc.) and mounted with coverslips. All washing steps were done three times with PBS-T (tween-20, 0.05%).