Ultravision lp detection system hrp dab kit

Sections of formalin-fixed, paraffin-embedded ascites and solid tumor patient-derived organoids were used for histopathological analyses. Organoids were recovered from BME using ice-cold cell recovery solution (Corning, New York, United States) in accordance with manufacturing protocols, fixed in phosphate-buffered 10% formalin, and embedded in 500 μL of Bio-Agar (Bio-Optica Milano Spa, Milano, ITA). Five μm sections were stained with hematoxylin and eosin (H&E) using a Leica ST5020 multistainer and 2 μm sections were cut for IHC analysis. The IHC was performed with an UltraVision LP Detection System HRP DAB kit (Thermo Scientific, Waltham, USA). Heat-induced antigen retrieval was performed using 10 mM citrate buffer pH 6.0. The following antibodies were used to characterize HGOC patients-derived organoids and parental tumors: PAX8 (ProteinTech Group, Germany, EU), WT1 (Abcam, U.K.), and CA-125 (Santacruz Biotechnology, TX).

Formalin-fixed, paraffin-embedded slides were deparaffinized in xylene and rehydrated in descending order of ethanol (100% to 70%). For antigen retrieval, Tris–EDTA (pH 9.0; Abcam; for CD31) or citrate buffer (pH 6.0; Abcam) was used according to the manufacturer’s instructions. The sections were incubated with primary antibodies (Additional file 1: Table S2) overnight at 4 °C. For chromogenic detection, an UltraVision LP Detection System HRP DAB kit (Thermo Fisher Scientific) was used according to the manufacturer’s instructions. After washing in PBST four times, reactivity was validated using a mouse/rabbit-specific HRP/DAB IHC Detection Kit (Micro-polymer, ab236466; Abcam). The slides were washed four times with distilled water and counterstained with Mayer's hematoxylin (4science, Gyeonggi-do, Korea) for 1.5 min at room temperature. After washing under running tap water, the slides were dehydrated in ascending order of ethanol (70%–100%). Images of representative fields were obtained using an Olympus BX43 light microscope (magnification, ×400; Olympus Corporation, Tokyo, Japan). Positively stained cells were quantified by calculating the mean number of positive cells from ten non-overlapping fields per slide at × magnification of ×400.