Histopathology, immunohistochemistry and confocal laser microscopy

Paraffin embedded tissue slides were examined histopathologically employing hematoxylin-eosin and Masson’s trichrome staining, and by immunohistochemical staining with specific endothelial and mesenchymal (myofibroblast) cell markers. The SSc and normal lung tissue samples were also examined by confocal laser microcopy as described previously (34). In all immunohistochemical and confocal laser microscopy studies samples incubated without primary antibody were used as negative controls. For immunohistochemistry the following primary antibodies were used: anti-CD31 (Neomarkers, Fremont, CA), anti-von Willebrand Factor (vWF, Dako, Denmark), anti-COL1 (Santa Cruz Biotechnology, Santa Cruz, CA), anti-COL3 (Fitzgerald, Acton, MA), and anti-α-SMA (Abcam, Cambridge, MA). For single antibody labeling, paraffin sections were immunoassayed by the peroxidase method using the indicated antibodies. For confocal laser microscopy paraffin samples were de-paraffinated and dehydrated following antigen retrieval with a citric acid buffer as described (34). Slides were first incubated with blocking IgG solution for 1 h and then overnight with one of the following antibodies: anti CD31 (1:50 dilution), anti α-SMA (1:200 dilution), anti-vWF (1:50 dilution), anti COL1 (1:200 dilution), or anti-COL3 (1:200 dilution) antibodies. IgG binding was revealed following incubation with (Fab′)-sheep anti-rabbit Cy3 antibody and (Fab′)-sheep anti-mouse FITC antibody (Sigma, St. Louis, MO) for 1 h. Nuclei were counterstained with 4,6-diamidino-2-phenylindole ( DAPI, Jackson ImmunoResearch Laboratories, West Grove, PA). Samples were examined with a Zeiss 51 confocal laser microscope (Zeiss, Thronwood, NY) to evaluate the co-localization of immunoreactivity with polyclonal and monoclonal antibodies in paired combinations of either CD31 or vWF with either α-SMA, COL1, or COL3.