Ultraab diluent

Paraffin embedding of formalin-fixed samples, sectioning, IHC, and slide scanning were performed by the Science for Life Laboratory facilities at the Department of Immunology, Genetics, and Pathology of Uppsala University, Sweden, as described [44 ]. Primary antibodies diluted in UltraAb Diluent (Thermo Fisher Scientific) were applied for 30 min at room temperature. The antibodies used at the indicated dilutions were: anti-GFP (Invitrogen A111222, 1:2000), anti-Ki67 (Abcam Ab15580, 1:1000), anti-Nestin (Millipore MAB5326, 1:100), anti-ID4 (Santa Cruz, sc-491, 1:100), anti-phospho-SMAD2 (Invitrogen 44-244 G, 1:200), and anti-PAI1 (Abcam, Ab66705 1:200). The slides were incubated with secondary antibody (Abcam Ab6721; Goat-Anti-Rabbit IgG H&L (horseradish peroxidase), 1:1000) for 30 min at room temperature and scanned using the automated scanning system Aperio XT (Aperio Technologies). Chromogen intensity was quantified using the reciprocal intensity (r) method [45 ], where: r = 255−y, 255 being the maximum pixel intensity of unstained area (as measured by the standard intensity function in the Fiji software) and y = mean intensity of the selected tumor area. Reciprocal intensity was quantified from one to six fields of three mice per group; the number of fields depended on the tumor size, and all analyses were performed without any blinding.