Patient data as well as blood samples of CM patients in Lübeck (CMPL) and Homburg (CMPH) were evaluated for this study after informed consent. A healthy individual’s blood sample was used as non-melanoma control (NMC). The blood was allowed to clot by leaving it undisturbed at room temperature (RT). The clot was removed and following centrifugation the resulting supernatant was collected as designated serum. Aliquots were stored at −80 °C. To verify the specific binding of antibodies in the sera, we used a commercially available unspecific human IgG (Perbio Science, Bonn, Germany) as antibody control (AC).
Frozen macaque (Macaca fascicularis) retina sections (8 µm) were thawed at RT and rehydrated in phosphate buffered saline (PBS). Demineralized paraffin cochlea sections (10 µm) from the same animals were deparaffinized and rehydrated in PBS as well. After applying blocking buffer, the slides were incubated with the different probes (AC, NMC, CMPL, CMPH1-10) for 2 h at RT. The slides were then repeatedly washed and incubated with a biotin-conjugated-goat-anti-human secondary antibody (Rockland, Gilbertsville, PA, USA) for 1 h at RT. After another washing procedure, slides were incubated with Alexa488-conjugated streptavidin (λ
em = 495 nm/519 nm; Life Technologies, Darmstadt, Germany) for 1 h and 4’,6-diamidino-2-phenylindole (DAPI λ
em = 358 nm/461 nm, 1 µg/ml Life Technologies, Darmstadt, Germany) for 10 min at RT. Finally, the slides were mounted using Mowiol mounting medium (Sigma Aldrich, Steinheim, Germany), and images were taken with an inverse fluorescence microscope (Leica DMI6000 B, Leica Microsystems, Wetzlar, Germany) using the same parameters (magnification, exposure time, contrast and saturation) for all specimens. Depending on the tissue (retina vs. cochlea), the same filter and threshold parameters were applied to all images before assessment by semi-quantitative measurement of the number of Alexa488-positive particles, using ImageJ software (Version 1.48b, NIH, Bethesda, USA) in a blinded fashion.
In addition, the NMC and MPL samples were examined for various cytokines (IFN-γ, IL1-13, IL17, IL23, CD14, CD163, TNFa) using a fully quantitative multiplex ELISA, according to manufacturer’s instruction (Q-Plex™ Human Cytokine Array, Quansys Biosciences, Logan, UT, USA). Data were captured on the Odyssey® infrared imaging system (Li-Cor Biosciences, Lincoln, NE, USA) and analyzed using Quansys Q-view Plus™ software (Quansys Biosciences, Logan, UT, USA).
All experiments were conducted thrice. Fold-changes relative to NMC are expressed as the mean ± standard deviation. Statistical analysis was performed with Prism (Version 6, GraphPad, La Jolla, USA) using an unpaired Student t test with p < 0.05 considered significant. This article does not contain any new studies with human or animal subjects performed by any of the authors.