196 Chapter 6 Immunohistochemistry Kryofix (50% ethanol, 3% PEG300)-fixed paraffin-embedded (KFPE) samples were cut into 5 µm thick sections and rehydrated followed by hematoxylin and eosin staining or blocking of endogenous peroxidase using 1% hydrogen peroxide for 15 min at RT. After antigen retrieval was performed (Supplementary Table 2), sections were pre-incubated with 5% normal goat serum (Abcam, Cambridge, UK) diluted in PBS + 1% bovine serum albumin. Sections were then incubated with primary antibodies for the detection of pan-cytokeratin, cytokeratin-10, cytokeratin-15, cytokeratin-17, involucrin, collagen IV, laminin α 5, vimentin, aSMA, Ki67, and BrdU (Supplementary Table 2) for 1 h at RT followed by incubation with a poly-HRP-goat-anti-mouse or rabbit secondary antibody (Bright Vision, VWR, Amsterdam, The Netherlands) for 30 min at RT. After washing, detection was established using 3,30-Diaminobenzidine (DAB). After DAB staining was completed, sections were counterstained with hematoxylin, dehydrated, and mounted with Eukit Mounting Medium (Sigma-Aldrich, St. Louis, MO, USA). For 5-bromo-20deoxyuridine (BrdU) staining, culture medium was supplemented with 20 µM BrdU (Sigma-Aldrich, St. Louis, MO, USA) 24 h before termination. Microscopy Microscopic visualization was performed with a Zeiss Axioskop40FL microscope (Zeiss, Breda, The Netherlands). Images were acquired using a Nikon Eclipse TS2 camera and the NIS-Elements software version 4.4 (Nikon Instruments, Amsterdam, The Netherlands). Re-epithelization rate Re-epithelization length was measured in microscopic images of H&E-stained sections using standardized measurement to calculate µm/pixel in NIS-Elements software. As both sides of each model were measured, the mean was used in the analysis. Immunoassay of culture medium Cytokines, chemokines, and growth factors were analyzed in samples of culture medium at T0, T + 1-4 days, T + 5-7 days, and T + 8-11 days (after burn injury). Samples from biological duplicates were pooled per donor (n = 3 donors). Neat samples were measured using the Human Essential Immune Response LegendPlex Multi-analyte Flow Assay kit (cat. 740929; BioLegend), according to the manufacturer’s instruction, and were acquired on a flow cytometer (MACS Quant Analyzer 10,Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). This 13-plex immunoassay included: IL-1β, IL-2, IL-4, IL-6, IL-8 (CXCL8), IL-10, IL-12p70, IL-17A, IP-10 (CXCL10), MCP-1 (CCL2), IFN-γ, TNF-α, and TGF-β1. Concentrations were determined using FlowLogic software (Inivai Technologies, Victoria, Australia) and recalculated to pg/mL per day of culture to compensate for differences in intervals of
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