167 Local Immune Response in Burn Patients Immunohistochemistry Kryofix (50% ethanol, 3% PEG300)-fixed paraffin-embedded 5 μm thick sections were used after deparaffinization and rehydration. Endogenous peroxidase was blocked in 1% hydrogen peroxide for 15 min at room temperature. Next, antigen retrieval for different antigens was performed. The blocking step was performed using 5% normal goat serum (Merck) diluted in PBS + 1% bovine serum albumin (BSA). Tissue sections were then incubated with the primary antibodies (Supplementary Table 3) for 1 h at RT followed by incubation with a poly-HRP-goat-anti-mouse or rabbit secondary antibody (BrightVision, VWR) for 30 min at RT. Detection of the target protein was established using 3,3′-Diaminobenzidine (BrightDAB, VWR). After immunohistochemical (IHC) DAB staining was successful, sections were counterstained with hematoxylin, dehydrated and mounted with Eukitt Mounting Medium (Merck). Percentage of MPO, CD3 or CD68 positive area was calculated using NIS Elements (Nikon Instruments Europe B.V.) and based on 3 images from representative tissue sections. Multiplex imaging and analysis Formalin-fixed and paraffin-embedded 5 μm sections were deparaffinized using xylene and rehydrated with ethanol and distilled water. Antigen retrieval was performed by boiling in TRIS-borate-EDTA buffer for 10 min. A multiplex staining for the detection of neutrophils and lymphocytes was performed performed using the protocol described by Rodriguez et al. [56]. Immunoassay of tissue homogenates Frozen tissue samples of approximately 60 mg were thawed, minced into smaller pieces and further dissociated in M-tubes (Miltenyi Biotec) by adding lysis buffer (PBS containing 0.01 mM EDTA and protease inhibitor (1 tablet per 10 mL; Pierce, Thermo Scientific)) and running program “Protein_01” on a gentleMACS (Miltenyi Biotec). Debris was removed from the samples using a filter plate (Multiscreen, Merck) and samples were diluted to concentration of 12 mg tissue/mL. Luminex assay was performed according to the manufacturer’s instructions (Merck KGaA). The following assay kits were used: HCYTA-60K, TGFBMAG-64K, HCYP2MAG-62K and HTH17MAG-14K. In short, 25 μL of tissue homogenate was used to determine the concentrations of 37 soluble factors, namely MCP-1 (CCL2), MIP-1α (CCL3), MIP-1β (CCL4), MIP-3α (CCL20), GROα (CXCL1), IP-10 (CXCL10), IFN-α2, IFN-γ, TNF-α, TGF-β1, TGF-β2, TGF-β3, CTACK (CCL27), RANTES (CCL5), IL-1α, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-8 (CXCL8), IL-9, IL-10, IL-12p40, IL-12p70, IL-13, IL-17A (CTLA-8), IL-17F, IL-18, IL-21, IL-22, IL-23, IL-33 (NF-HEV), GM-CSF, PDGF-AA, PDGF-AB/BB and VEGF-A. For TGF-β1,2,3 samples were acid-treated prior to the assay, according to the manufacturer’s instructions. Mean fluorescence intensity of samples was measured with a Flexmap 3D System (Luminex Corp, Austin, USA) and concentrations were calculated 5
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