We performed immunohistochemistry as described previously [11 ]. In brief, we incubated slides with αPCNA, (1:100; Santa Cruz, CA); or αSEMA5A [5 (link)] (1:50) primary antibodies in antibody diluent overnight at 4 °C. Next morning, we washed the slides and incubated with biotinylated anti-rabbit or anti-mouse secondary antibody (Vector Laboratories, Burlingame, CA) for 45 min. We quantitated the cell number for a particular stain by counting at least five different random fields in the same section at high resolution using a Nikon microscope.
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