Sigmafast dab metal enhancer

Human ADPKD and NHK renal tissue sections were deparaffinized in xylene and rehydrated through an ethanol series to distilled water. Antigen retrieval was performed by steaming tissue sections for 25 minutes in Sodium Citrate Buffer (10 mM Sodium Citrate (Fisher Scientific), 0.05% Tween 20 (Fisher Scientific) in autoclaved water, pH 6.0). To minimize background staining, sections were treated with 3% hydrogen peroxide for 30 min, washed in PBS, then blocked with 1% BSA for 1 hour. Cells were then incubated with GLI1 antibody (Cell Signaling) overnight at 4 °C. Following 3 washes in PBS, sections were incubated with HRP-conjugated rabbit secondary antibody (Cell Signaling) for 30 minutes. Following another 3 washes in PBS, tissues were incubated with ABC reagent (Vector Laboratories), rinsed in PBS, and then incubated with SigmaFAST DAB metal enhancer (Sigma) until desired signal/color was obtained. To determine GLI1 localization in proximal tubules or collecting ducts, sections were incubated with fluorescein-conjugated Lotus tetragonolobulus or Dolichus biflorus agglutinin for 1 hour at room temperature, washed and mounted in Vectashield containing 4,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories). Staining was visualized and imaged using a Nikon 80i light/fluorescent microscope and Nikon DS-Fi1 camera. Immunohistochemistry images were converted to grayscale.