For immunohistochemical detection, 5 μm paraffin sections were used; and endogenous biotin and peroxidase activity were blocked. The color was developed with DAB chromogen (ImmPACT DAB; VectorLabs, United States) and the sections were counterstained with hematoxylin.
Immunofluorescence analysis was performed as described22 (link). 6~8 μm frozen backskin sections were fixed in 4% PFA. For blocking, the MOM kit (Vector Laboratories, Burlingame, CA) was used for mouse monoclonal antibodies, and the following blocking buffer for all other antibodies: 10% normal donkey serum, 2% BSA, 2% fish skin gelatin, 2% Triton X-100 in PBS. Primary antibodies were used at the following concentration: guinea pig anti-Tcf3 (1:200, lab-generated), rabbit anti-Tcf4 (1:200; Cell Signalling), rabbit anti-Tcf1 (1:50; Cell Signaling), rabbit anti-Lef1 (1:150; Cell Signaling), mouse anti-myc tag (1:500; Zymed), mouse anti-β-catenin (Sigma), mouse anti-Stat3 (1:300; Cell Signaling), pY705-Stat3 (1:100; Cell Signaling), rat anti-cd104 (1:200; BD Biosciences) goat anti-Lcn2 (1:150;R&D Systems). Antigens were visualized with FITC-or RRX-conjugated secondary Abs (Jackson Labs). All images were acquired with Zeiss Axioskop microscope.
Immunofluorescence analysis was performed as described22 (link). 6~8 μm frozen backskin sections were fixed in 4% PFA. For blocking, the MOM kit (Vector Laboratories, Burlingame, CA) was used for mouse monoclonal antibodies, and the following blocking buffer for all other antibodies: 10% normal donkey serum, 2% BSA, 2% fish skin gelatin, 2% Triton X-100 in PBS. Primary antibodies were used at the following concentration: guinea pig anti-Tcf3 (1:200, lab-generated), rabbit anti-Tcf4 (1:200; Cell Signalling), rabbit anti-Tcf1 (1:50; Cell Signaling), rabbit anti-Lef1 (1:150; Cell Signaling), mouse anti-myc tag (1:500; Zymed), mouse anti-β-catenin (Sigma), mouse anti-Stat3 (1:300; Cell Signaling), pY705-Stat3 (1:100; Cell Signaling), rat anti-cd104 (1:200; BD Biosciences) goat anti-Lcn2 (1:150;R&D Systems). Antigens were visualized with FITC-or RRX-conjugated secondary Abs (Jackson Labs). All images were acquired with Zeiss Axioskop microscope.