Fluorescent dyes

For immunohistochemical (IHC) staining, deparaffinized and hydrated lung and placental tissue sections were washed in 0.05% v Brij-35 in PBS (pH 7.4) and immunostained for antigen expression as described previously (41 (link)). Briefly, the antigens were unmasked by steaming the sections in 10 mM Citrate buffer (pH 6.0) followed by incubation in a blocking solution containing 3% BSA, 1% Gelatin and 1% normal donkey serum with 0.1% Triton X-100 and 0.1% Saponin. Serial sections were stained with antibodies to Vimentin, E-cadherin, and ZO-1 (Invitrogen Inc., Carlsbad, CA), or isotype control antibodies. The immunolabelled tissues were detected using respective secondary antibodies conjugated with fluorescent dyes (Jackson ImmunoResearch Lab Inc., West Grove, PA). Where indicated, the sections were stained with 4′,6-diamidino-2-phenylindole (DAPI) containing Fluormount-G (SouthernBiotech, Birmingham, AL) to visualize nuclei. Immunofluorescent images were captured with BZX700 Microscopy system (Keyence, Tokyo, Japan). Specific details are given under appropriate figure legends.

After washing with 0.01 M phosphate buffered saline (PBS) for 10 min, sections were incubated in blocking solution (5% BSA and 0.3% Triton X-100 in 0.01 M PBS) for 1 hr at room temperature. Then the sections were incubated in primary antibodies (rabbit anti-Iba-1, lot 019–19741, 1∶1000, Wako Chemical) overnight at 4°C. On the second day, these sections were washed with 0.01 M PBS for three times and then incubated in the secondary antibodies labeled with fluorescent dyes (1∶200, Jackson Immunoresearch) for 2 hours at room temperature. Through three washes of PBS, these sections were covered with mounting medium with DAPI (vector). As negative controls, an adjacent series of sections were processed using the same procedures without the primary antibodies.