Streptavidin peroxidase solution

The secretion of IL-8 in differentiated HepaRG cells following exposure to C17-SAMT for 24 h was measured by ELISA.
Briefly, plates (Maxisorp NUNC44240) were coated with the capture antibodies (AC IR Human IL8 Mab, M801, ThermoFisher, Grigny, France) at 4 °C overnight and then washed 3 times with 100 μL/well PBS-0.05% Tween-20 buffer. Coated plates were blocked with 100 μL of a Super Block Buffer blocking solution (Thermofisher, 37515) for 1 h at room temperature. After washing three times, 90 μL of the diluted medium was added to each well and incubated at RT for 90 min with continuous agitation, followed by three washing steps. Wells were then incubated with 50 μL of the diluted secondary biotinylated antibodies (IL-8 Biotinylated, ThermoFisher, M802) for 1 h, followed by three washes with PBS-Tween. For spectrophotometric detection, wells were incubated with 100 µL of a streptavidin peroxidase solution (ThermoFisher, ref21132). Following washes, plates were incubated with 100 μL/well 3,3′,5,5′-Tetramethylbenzidine TMB (ThermoFisher, 34028) for 45 min at RT. The reaction was terminated by the addition of 50 μL/well 1 M H₂SO₄. The absorbance at 405 nm was quantified using a Fluostar Optima^® plate reader (BMG Labtech, Elancourt, France).

After deparaffinization and rehydration using xylene and ethanol, sections (4 mm thick) from paraffin-embedded tumor tissues were immersed for 10 min in 3% hydrogen peroxide solution (Sigma-Aldrich, St. Louis, MO, USA). After rinsed with PBS, sections were added with primary antibodies (1:500), secondary antibodies (1:2000), streptavidin-peroxidase solution (S-P, Thermo fisher), and diaminoaniline (DAB, # ab64261, Abcam, Cambridge, MA, USA) for 3-5 minutes. Counterstained with hematoxylin (Beyotime, Jiangsu, Shanghai), sections were observed under an optical microscope (Olympus, Tokyo, Japan).