Adenoviral-Mediated Skin Tissue Analysis

Human Samples: Human skin was obtained from corrective plastic surgery. All tissues were obtained according to the guidelines of the University of Pittsburgh and under a protocol approved by the Institutional Review Board of the University of Pittsburgh. Subcutaneous fat tissue was removed and skin tissue was cut into 1.5 cm x 1.5 cm sections. Adenoviral (Ad) constructs were injected intradermally in a volume of 100 µl 1x PBS. Explants containing complete epidermal and dermal layers were cultured in an air liquid interface with the epidermal and keratin layers side up and exposed to air. The culture medium was replaced daily and consisted of Dulbecco’s modified Eagle’s medium (DMEM) (Mediatech, Herndon, VA) supplemented with 10% FBS (Sigma-Aldrich, St Louis, MO), penicillin, streptomycin, and anti-mycotic agent (Invitrogen Life Technologies, Carlsbad, CA). At the indicated time points, skin tissue was harvested and fixed in 10% formalin prior to embedding in paraffin. Skin punch biopsies were obtained from the clinically affected and unaffected skin of patients with SSc as we have previously described [9 (link), 11 (link)].
Adenoviral Constructs: Replication deficient adenoviruses serotype 5 encoding human IGFBP-3, IGFBP-4, or IGFBP-5 were generated as previously described [10 (link)]. Adenovirus serotype 5 lacking cDNA was used as a control. Adenoviruses (1 x 10⁸ pfu) were injected intradermally in a 100 µl volume.
Immunohistochemistry (IHC): Six µm sections of paraffin embedded tissues were deparaffinized and endogenous peroxidases were quenched with 3% H₂O₂. Sections were blocked with 5% serum and incubated with polyclonal anti-IGFBP-5 antibody (Gropep Ltd, Adelaide, Australia) or IgG control antibody (Lab Vision Corporation, Fremont, CA). Sections were washed and incubated with biontinylated secondary antibody (Vector Laboratories, Burlingame CA). Bound secondary antibody was detected using the Vectastain ABC kit (Vector Laboratories) and Zymed AEC Red kit (Zymed, San Francisco CA). A light hematoxylin counterstain was used to identify nuclei using Hematoxylin QS (Vector Laboratories). Images were taken on a Nikon Eclipse 800 microscope (Nikon Instruments Inc., Huntley, IL) using identical camera settings.
Measurement of Skin Dermal and Collagen Bundle Thickness:Six µm sections of paraffin-embedded skin tissue were stained with hematoxylin and eosin (H & E). Images were taken on a Nikon Eclipse 800 microscope. The thickness of the dermis and of individual collagen bundles was measured using Microsuite™ Software (Olympus America Inc.) as we previously described [11 (link)]. Thickness was measured in 5 random fields in each sample. Data are shown in arbitrary units.
Statistical Analysis:Dermal and collagen bundle thickness were analyzed using the Mann-Whitney U test.