Immpact dab diluent

Paraffin-embedded lung sections were deparaffinized and rehydrated with Citrisolv (Thermo Fisher Scientific, Waltham, MA) and graded ethanol, treated with antigen retrieval solution (Vector Lab, Burlingame, CA), then incubated with 3% hydrogen peroxide, and blocked with 2.5% horse serum in Tris-buffered saline and Tween 20 (TBST) prior to incubation with primary antibodies. For dual fluorescent staining, sections were incubated overnight with mouse monoclonal antimouse α-smooth muscle actin antibody (α-SMA, 1:100, clone 1A4; Sigma, St. Louis, MO) and rabbit polyclonal von-Willebrand Factor (FVIII, 1:500; Sigma, St. Louis, MO). Next day, sections were treated with Alexa 594 (1:500) and Alexa 488 (1:2000) fluorescent secondary antibodies (Invitrogen, Carlsbad, CA). Slides were counterstained with methyl green (Vector Laboratories, Burlingame, CA) to stain the nuclei. Additional slides stained for α-SMA were incubated with an anti IgG antibody followed by the ABC reagent (Vectastatin kit; Vector Laboratories, Burlingame, CA), developed with ImmPact DAB diluent (Vector Laboratories, Burlingame, CA) and counterstained with hematoxylin for morphometric analysis. Tissue sections were examined by light microscopy and photographed on a Zeiss Axiovert S100 (Carl Zeiss LLC, Thornwood, NY) at 100× magnification, and images were analyzed using AxioVision software.

Immunohistochemistry was performed using the mouse monoclonal α-smooth muscle actin (α-SMA) antibody (1:1000, clone1A4; Sigma, St. Louis, MO) and MOM kit with biotinylated anti-mouse secondary IgG antibody, per instructions (Vector Laboratories, Burlingame, CA), as well as rabbit anti-human/mouse Factor VIII) (1:1000, Sigma), with biotinylated anti-rabbit secondary IgG antibody (1:100; Vector Laboratories). Slides were developed with ImmPact DAB diluent (Vector Laboratories) and counterstained with hematoxylin.