Fitc conjugated donkey anti mouse

Sources of primary antibodies used in these studies are listed in Table S2. Five μm frozen sections cut from optimal cutting temperature-embedded tissues were washed in PBS three times for 5 min each, then incubated in blocking buffer (3% BSA/0.1% Tween-20) for 30 min. Next, the sections were incubated for 1h with a primary antibody diluted 1:100 in blocking buffer. These antibodies included anti-Rac1 (Millipore, cat. #05-389), anti-S100A7 (Imgenex cat. # IMG-409E), anti-SSFA2 (Sigma, cat. #SAB1100788), anti-IGFBP5 (R&D systems, cat. #AF875), anti-USP16 (Abcam, cat. #ab121650), and an anti-cytokeratin (CK7) developed in the Fisher lab ^{30 (link)}. Tissue sections were washed and incubated with species-specific secondary antibodies for 30 min. Secondary antibodies (Jackson Immunoresearch) included TRITC-conjugated donkey anti-rat (prod. #712-026-153), FITC-conjugated donkey anti-mouse (Jackson Immunoresearch cat. #715-095-151), FITC-conjugated donkey anti-goat (cat. # 705-096-147), and FITC-conjugated donkey anti-rabbit (cat. #711-096-152). Then the tissue sections were washed, rinsed in dH₂O, dried, and mounted using Vectashield Mounting medium with DAPI (cat. #H-1200). Immunoreactivity was imaged using a Leica DM 5000B fluorescence microscope equipped with a Leica DFC 350FX digital camera (Leica Instruments). Immunoblotting was performed as previously described^{31 (link)}.