Vectashield mounting medium

For mRNA imaging, cells were fixed with 4% paraformaldehyde (PFA) for 20 min at room temperature (RT) after transfection for 24 h, permeabilized in 70% ethanol at 4°C for 1 h and mounted in Vectashield mounting medium (Vector Laboratories) with DAPI. Cell imaging was performed using an Olympus FV3000 confocal microscope. For immunocytochemistry in neurons, cells were fixed with 4% PFA for 20 min at RT. Permeabilization was performed in 0.1% Triton X-100 in PBS for 10 min at RT and blocked for 1 hour in PBS containing 3% bovine serum albumin (BSA). After blocking, cells were stained for primary antibodies: anti-Myc (1:1,000, Abcam, ab9132), anti-DYKDDDDK (FLAG) Tag (1:1,500, Cell Signaling Technology, 8146), anti-MAP2 (1:300, Millipore, 05-346), anti-HA tag (1:200, Abcam, ab9110) overnight at 4 ºC in blocking buffer, followed by labeling with corresponding fluorescent secondary antibodies (1:500, Invitrogen) for 2 h at RT in blocking buffer. After staining with DAPI, cells were washed and mounted in Vectashield mounting medium (Vector Laboratories).

Frozen tumor tissue slices of 5 mm thickness were fixed with cold acetone for 10 min, washed with PBS, and then, blocked with 10% donkey serum for 45 min at room temperature. Slices were then incubated overnight with pertuzumab (ThermoFisher Scientific, USA) at 4°C [16 (link)]. Next, sections were further stained with AlexaFluor488-labeled goat anti-human antibody (ThermoFisher Scientific, USA). A coverglass was applied to each slide using Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA), and imaging was performed using an A1R confocal microscope (Nikon, Nikon Instruments, Melville, NY, USA).

Cryopreserved and decellularized tracheal tissues were compared to native trachea by a fluorescence analysis based on 4′,6-Diamidino-2-Phenylindole (DAPI) aiming to assess nuclei presence/distribution. The epithelial layer, the mucosa, and submucosa layers and the cartilaginous compartments were all considered, for a full-thickness description of the tissue. Briefly, small tissue samples were excised, Optimal Cutting Temperature (OCT) medium embedded and frozen prior to being cut into 5 µm thick sections using a cryomicrotome (Leica CM 1850 UV; Leica Microsystems, Wetzlar, Germany); hence, the sections were fixed with acetone, mounted with Vectashield mounting medium for fluorescence with DAPI (Vector Laboratories, Burlingame, CA, USA) and photomicrographs were acquired with a Leica LMD6 (Leica Microsystems) connected to a Leica DFC320 high-resolution digital camera (Leica Microsystems) and a computer equipped with software for image acquisition (LasX, Leica Microsystems).

Patients and healthy individuals’ blood samples were split into three samples. One sample was used as control to detect the spontaneous γH2AX foci formation. The second sample was ex vivo irradiated with 0.5 Gy X-rays and 30 minutes incubation time and the third one with 2 Gy and 24 hours incubation time. The two different doses were chosen, because a dose of 2 Gy induces after 30 min such a high number of foci that it is not possible to count the foci accurately. On the contrary, with a dose of 0.5 Gy and 24 hours repair the amount of foci is too low to have sufficient foci numbers. Therefore for the initial γH2AX foci a low and for the remaining γH2AX foci after 24 hours repair time a high dose was chosen. Peripheral blood mononucleated cells (PBMC) were isolated by Ficoll gradient centrifugation and were cytocentrifuged (StatspinCytofuge, Kreatech, Germany) onto a specimen. The samples were fixed with methanol and acetone and afterwards washed in a phosphate-buffered saline with foetal calf serum. The slides were then incubated with a mouse anti-γH2AX antibody (Abcam, Cambridge, UK), washed in PBS and incubated with a secondary goat anti-mouse Alexa 488 fluorescent antibody (Molecular Probes, Karlsruhe, Germany). Afterwards lymphocytes were washed in PBS and mounted with Vectashield mounting medium (Vector Laboratories, Peterborough, UK).

HBL-H2BGFP melanospheres were cultured for 7 days in the presence or absence of TNF (0.5 μg/ml) and/or LY294002 (10 μM), which were both added at seeding. The dissociated melanosphere cells were plated on Lab-Tek chamber glass coverslips (Millicell EZ SLIDE 8-well glass, sterile Merck Milipore, Darmstadt, Germany) at a density of 15 000 cells per well. After 24 hours, the adherent cells were fixed in PAF solution, and immunocytochemistry was performed according to the standard procedure. A monoclonal anti-Melan A antibody, which was purchased from Santa Cruz Biotech, was used at a dilution of 1:100, and positive cells were detected with a secondary AlexaFluor 594 goat anti-mouse (Life Technologies), which was used at a dilution of 1:2000. Negative controls were performed by replacing the primary antibody with an irrelevant isotype. Nuclei were counterstained with DAPI. All slides were mounted under a coverslip with Vectashield mounting medium (Vector Laboratories, Nanterre, France) and were photographed using a Leica DMRB LAS3.7 fluorescence microscope.