Protein analyses

4.5.1 Immunofluorescence analyses

Cells grown on glass chamber slides were fixed with 4 % paraformaldehyde in PBS for 15 min and permeabilized with 0.1 % Triton X-100 in PBS for 10 min. Cryosections (6 µm thick) of human skin and organotypic 3-D skin models were dried on slides for 1 h and fixed with ice–cold acetone for 10 min. To block unspecific binding sites, cells and sections were incubated in 1 % BSA in PBS for 1 h. Prior to staining, specificity of the customized OXTR-directed antibody SYC592 was validated by performing a blocking peptide experiment. The blocking peptide was designed to mimic a specific epitope of the OXTR recognized by SYC592. The antibody SYC592 was pre-incubated with a 20-fold excess by weight of the blocking peptide for 1 h leading to its neutralization. In the following staining procedure, the neutralized antibody was used like the primary antibody. Incubation with primary antibodies (Table 1) in 1 % BSA in PBS was performed at 4 °C overnight. For visualization and staining of the nuclei, cells and cryosections were incubated with fluorophore-conjugated secondary antibodies mixed with DAPI (Table 1) in 1 % BSA in PBS for 1 h. An Axiovert-S100 microscope and the AxioVision 4.8 software were used for immunofluorescence analyses.

4.5.2 Measurement of epidermal layer thickness

After immunohistochemical staining of the organotypic 3-D skin models, their epidermal layer thickness was analyzed. For this purpose, each boundary between the stratum corneum, the cytokeratin 5/14- and the cytokeratin 1/10-positive layer was manually traced utilizing the Adobe Photoshop CS6 software. Subsequently, the average space between these layers throughout the entire section was calculated by using an internal program of the Beiersdorf AG (Figure 10). A total of 6 sections derived from different parts of each 3-D skin model were analyzed.

Figure 10. Tracing of epidermal layers of a 3-D skin model.

(a) Immunohistochemical staining of epidermal layers of a 3-D skin model section with antibodies directed against human cytokeratin 5/14 (green) and cytokeratin 1/10 (red). (b) The boundaries of the layers were traced. As an example, tracing of the cytokeratin 5/14-positive layer is depicted (yellow lines). (c) The average space between these boundaries over the entire section was calculated by using an internal program of the Beiersdorf AG.

4.5.3 Cell lysis and preparation of membrane fractions

Frozen pellets of dermal fibroblasts were subjected to lysis and extraction of membrane fractions using the ProteoExtract^® Subcellular Proteome Extraction Kit. Afterwards, protein concentrations were determined via BC Assay (4.5.4).

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4.5.4 Quantification of protein concentrations

Protein concentrations of membrane fractions were determined by utilizing the BC Assay Protein Quantification Kit. This colorimetric assay is based on a variant of the Biuret reaction.

Under alkaline conditions, Cu²⁺ coordinates 4 peptide bonds leading to its reduction to Cu⁺. Two bicinchoninic acid molecules chelate Cu⁺, forming a violet coloured complex that absorbs at 562 nm. The absorbance is directly proportional to the protein concentration and was detected using a Safire photometer. Protein concentrations were calculated with a standard curve obtained for bovine serum albumin.

4.5.5 SDS-Polyacrylamide gel electrophoresis

Proteins were separated according to their relative molecular mass under denaturing conditions by performing a sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). SDS molecules bind to proteins to linearize them and to impart an even distributed negative charge per mass. Additionally, ß-mercaptoethanol is used to disrupt tertiary structures of proteins by oxidizing disulfide bonds. Thus, during electrophoresis the migration of proteins correlates with their approximate molecular weight.

The protein samples (4.5.3) were supplemented with 2x Laemmli loading buffer containing 5 % (v/v) ß-mercaptoethanol and boiled for 8 min at 99 °C. Both, the MagicMark™ XP Western Protein Standard and the Novex^® Sharp Pre-Stained Protein Standard, were used as molecular weight markers. Samples were loaded on a 4-15 % Criterion Tris-HCl gradient Gel inserted in a Criterion™ electrophoresis cell. After filling the gel chamber with running buffer (Table 13), a constant voltage (120 V) was applied until the loading dye front arrived at the bottom of the gel. Finally, the separated proteins were subjected to western blot analysis.

4.5.6 Western Blot

Western blotting was carried out to allow immunological detection of the OXTR. For this purpose, the via SDS-PAGE (4.5.5) separated proteins were transferred onto a PVDF membrane in a Criterion™ wet blot tank. Prior to blotting, the membrane was briefly equilibrated in methanol. An air-bubble-free “blot sandwich” was arranged, consisting of membrane, gel, filter paper and sponges soaked with blotting buffer (Table 13). The Blot sandwich was inserted into the tank filled with blotting buffer. Subsequently, this aparatus was placed in an ice-bath to prevent heating. Then western blotting was conducted at a constant current of 750 mA for 1 h. All following incubation steps took place on a shaker. For immunodetection, unspecific binding sites were blocked with 5 % (w/v) skim milk powder in TBS for 1 h. Afterwards, the membrane was incubated with a primary antibody (Table 1) in 5 % (w/v) skim milk powder in TBS at 4 °C overnight. After washing 3 times for 10 min in

TBS-T, the membrane was incubated with a fluorescent dye-labeled secondary antibody (from Li-cor) (Table 1) in 5 % (w/v) skim milk powder in TBS for 1 h. The washing step was repeated thrice and immunofluorescence was detected using the Odyssey Infrared Imaging System.

4.5.7 Detection of oxytocin protein levels

OXT concentrations in suction blister fluids and cell culture supernatants were measured using an OXT-ELISA and normalized to total protein concentrations measured by BC quantification (4.5.4).

4.5.8 Detection of cytokine protein levels

Cytokine concentrations of cell culture supernatants were measured using a 27-Plex Panel (IL1β, IL1rα, IL2, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL12, IL13, IL15, IL17, FGF, Eotaxin, G-CSF, GM-G-CSF, IFNγ, CXCL10, MCP1, MIP-1α, MIP-1β, PDGFbb, CCL5, TNFα, VEGF).

The Bio-Plex technique permits the simultaneous detection and quantification of multiple cytokines in a single well of a 96-well microplate. The principle of the assay is similar to a capture sandwich immunoassay in suspension form. The capture antibody is immobilized on a magnetic bead and the final measurement is performed by flow cytometry.

Antibodies directed against a specific cytokine are covalently coupled to fluorophore-conjugated beads. The specific color signature of the beads is based on specific amounts of two distinct fluorophores. Thus, each cytokine corresponds to a different color of the beads allowing the parallel detection of up to 100 cytokines in the same sample. When the beads are incubated with the sample, the protein of interest is captured and then a biotinylated antibody for a different epitope is added to the reaction, which is then detected with streptavidin-phycoerythrin. Using a dual-laser reader, beads are analyzed for the color signature of the bead and the biotinylated detection antibody, identifying both the protein analyzed and the quantity bound to the bead.