P smad2 s465 467 antibody

Four hours following the incubation of Sca1⁺ cells with PKH67⁺ EVs (10⁹ particles per 4 × 10⁵ cells) in the StemMACS media (Miltenyi Biotec), cells were stained with cell surface markers (SLAM). Cells were fixed and permeabilized with BD Cytofix/Cytoperm Plus Fixation/Permeabilization Kit and Permeabilization Buffer Plus (BD Biosciences). We used the p-Smad2 (S465/467) antibody (#18338, Cell Signaling Technology) and the secondary anti-Rabbit-AF568 antibody (Thermo Fisher Scientific). Then, SLAM cells were purified by FACS and applied on a glass slide (Superfrost plus, Thermo Fisher Scientific) for up to 10 min and fixed with ProLong Gold Antifade reagent containing DAPI (P36931, Thermo Fisher Scientific). Fluorescent images were acquired with an Axio Imager M2 (Zeiss). Fluorescent optical sections of cells were obtained under magnification ×63 using an Axio Imager M2 (Zeiss) coupled with an Apotome.2 (Zeiss). Fluorescent images were processed for study (Fiji, NIH software).

Liver tissues were fixed in 4% buffered formalin solution and embedded in paraffin. Four-μm sections were stained with H&E or processed for immunohistochemistry. Antigen retrieval was performed by microwave treatment in EDTA buffer (1 mM, pH 8.0). Slides were blocked with peroxidase blocking reagent (Dako, Hamburg, Germany) for 30 min and 10% H₂O₂ for 15 min at room temperature. Then slides were washed with PBS and incubated with the SMAD7 antibody (1:100; ZB-8, Santa Cruz, Dallas, TX, USA), p-STAT3 antibody (1:100; 9145, Cell Signaling Technology, Danvers, MA, USA), pSmad2(S465/467) antibody (1:100, Cell Signaling Technology, 3101L), Ki67(D3B5) antibody (1:50, Cell signaling Technology, 12202S) or P21WAF1/Cip1 antibody (1:100, Sigma, P1484) at 4 °C overnight. Slides were washed with PBS twice, incubated with streptavidin-conjugated horseradish peroxidase antibody for 30 min and developed with diaminobenzidine (Sigma Aldrich, Munich, Germany) or for pSTAT3 stainings using the EnVision Detection Systems (K4065, Dako). Then slides were washes and counterstained with hematoxylin. Immunoreactivity was examined under a light microscope. Sections treated with secondary antibodies only were used as negative control.