Distribution of proliferating cells in normal and dcSSc skin. Cryosections from (a) normal and (b,c) dcSSc were stained with an anti-PCNA antibody. In normal skin, PCNA immunostaining was restricted to cells within the epidermis and sweat glands ((a,b) arrows). In two out of ten dcSSc samples, PCNA was detected in fibroblastic cells ((b) arrows) and in microvessels ((c) arrows). Double immunofluorescence labelling of dcSSc skin: cryosections were double stained with a combination of antibodies against (d,e) PCNA and α-SMA and (f) PCNA and PAL-E. PCNA is labelled with Texas Red while α-SMA and PAL-E are labelled with FITC. Colocalization was detected with PCNA and α-SMA antibodies within the microvasculature ((d,e) arrows, yellow colour). When used in combination with PAL-E, PCNA-labelled cells ((f) arrows) were predominantly located adjacent and abluminal to endothelial cells ((f) arrowheads). Original magnification × 20. α-SMA, alpha smooth muscle actin; dcSSc, diffuse cutaneous systemic sclerosis; PCNA, proliferating cell nuclear antigen; FITC, fluorescein isothiocyanate.