Aperio cs2

Liver tissues were embedded in paraffin, sliced in 5 μm sections and deparaffinized with Xylene. For H&E stainings sections were rehydrated, washed with PBS and counterstained with Harris Hematoxylin (1:10 Roth, Newport Beach, CA, USA) before being rinsed with acid water followed by 10 min wash with tap water. Sections were incubated with eosin for 5 min, shortly rinsed, dehydrated and mounted with DPX mounting medium (Sigma-Aldrich, Belgium). For Collagen 4 staining, sections were rehydrated, washed with PBS-0.05%Tween (PBST) and endogenous peroxidase was quenched with 3% H₂O₂ in methanol. Samples were washed three times with PBST for 5 min and incubated with 2% BSA-PBS for 1 h at room temperature. Col4 antibody (2 μg/ml, ab6586, Abcam, UK) was dissolved in 1% BSA-PBS and incubated overnight at 4°C. Sections were washed and incubated with Dako EnVision+ System- HRP Labeled Poly (K4003, Dako, Denmark) for 30 min at room temperature. Sections were washed with PBST, incubated with DAB substrate for 3 min at room temperature. Finally, samples were rinsed, counterstained with Harris Hematoxylin (1:10) and mounted with DPX mounting medium (Sigma-Aldrich, Belgium) and imaged visualized with Leica Aperio CS2 (Leica, The Netherlands). Quantification was performed with Orbit image analysis (31 (link)).

Female SD rats at 3 weeks of age with bodyweights ranging from 62.3 to 82.3 g were randomized into three groups of 8 animals each and treated with vehicle, tamoxifen (60 mg/kg), or SCR-6852 (10 mg/kg) by oral gavage once daily for three consecutive days. Twenty-four hours after the final dose, all animals were euthanized. Body weights and wet uterine weights were recorded for each animal. Fresh uterine tissue from each rat was fixed in 4% paraformaldehyde, dehydrated by HistoCore PEARL (Leica), and embedded by HistoCore Arcadia H + HistoCore Arcadia C (Leica). Sections were cut at 4 μm and stained with 0.1% toluidine blue O. The thickness of endometrial epithelium was measured using Leica Aperio CS2 with ImageScope × 64 program (Leica). The mean of five measurements per specimen was calculated.

TUNEL staining was conducted using the ApopTag kit (Oncor, Purchase, NY, United States). Paraffin sections were dewaxed and repaired with proteinase K and then incubated with TdT enzyme and d UTP for 2 h at 37°C while nuclei were stained with DAPI. Images were scanned and analyzed using Aperio CS2, Leica.