Double immunofluorescence labelling of normal and dcSSc skin biopsies. Cryosections from (a,c) normal and (b,d) dcSSc were double stained for endothelial cells using (a,b) PAL-E antibody and Thy-1 and (c,d) α-SMA and Thy-1. Thy-1 is labelled with FITC while PAL-E and α-SMA are labelled with Texas Red. In both (a) normal and (b) dcSSc, immunofluorescence for Thy-1 ((a,b) arrow, green colour) and PAL-E ((a,b) arrowhead, red colour) was consistently exclusive and showed no colocalization. In both (c) normal and (d) dcSSc, strong colocalization between Thy-1 and α-SMA was evident ((c,d) arrows, yellow colour). In normal skin, Thy-1 immunofluorescence that did not colocalize with α-SMA was observed immediately adjacent to microvessels ((c) arrowheads, green colour). Cryosections from dcSSc were double stained for (e,f,g) ED-A FN and α-SMA and (h) ED-A FN and Thy-1. ED-A FN is labelled with Texas Red while α-SMA and Thy-1 are labelled with FITC. Cell nuclei are counterstained blue with DAPI. Colocalization between α-SMA and ED-A FN was detected in dermal fibroblastic cells ((e) arrows, yellow colour) as well as in the microvascular wall ((f,g) arrows, yellow colour). Colocalization was also observed between ED-A FN and Thy-1 in both the microvascular wall ((h) arrow, yellow colour) and in dermal fibroblastic cells ((h) arrowheads, yellow colour). Original magnification (a-d,h) × 10, (e,f) × 20, (g) × 40. α-SMA, alpha smooth muscle actin; DAPI, 4,6-diamidino-2-phenylindole; dcSSc, diffuse cutaneous systemic sclerosis; ED-A FN, ED-A splice variant of fibronectin; FITC, fluorescein isothiocyanate.