Leica sp8 microscope

Histological examination was performed on 6 μm sections stained with hematoxylin-eosin safran (HES) and with Sirius red. Microscopic examinations were done with a x10 and x20 objectives lens using Vectra^TM system or with a x40 objective lens using confocal SP8 Leica microscope.

We scanned the ISH and ISH/IHC images at high resolution with the Aperio ImageScope software (Leica Biosystems). Immunofluorescent (IF) images were obtained from the sectioned brains and the whole-mounted explants using a confocal SP8 Leica microscope. Individual optic sections were 3 µm apart, and image stacks of various Z sizes were generated according to the structures of interest. All figures from the Allen Developing Mouse Brain Atlas (https://developingmouse.brain-map.org/) were 180º rotated (nose at the right side) to have the same orientation than in our own lab images. Figures were constructed using ImageJ, Adobe Photoshop and Adobe Illustrator software.

To quantify the phagocytic capacity of HMC3, cells were grown in glass bottom chamber slides. After treatment, cells were incubated with 5 µl of prelabeled zymosan particles (ab234054, Abcam) for 2 h and then washed by adding cold phagocytosis assay buffer. Cells were analyzed by confocal fluorescent microscopy (SP8 Leica microscope).

K562 cells were transfected by nucleofection (Amaxa Nucleofector Technology, Lonza AG) using a GPI-GFP plasmid (kind gift from D. Davis). After 24 h cells were sorted based on GFP fluorescence and co-cultured with HS-5 cells on Lab-Tek Chamber Slides (ThermoFisher Scientific) for 48 h. Additionally, both cell types were stained with WGA-Alexa Fluor 647 (ThermoFisher Scientific). Images were acquired using an SP8 Leica microscope with a ×63 objective and analyzed using ImageJ software.

Cells were seeded and grown in multi-well 24 plates until reaching 80% of confluence. Cells were incubated with 1 µM of FluoZin-3AM (Invitrogen, Darmstadt, Germany) or 25 µM of Zinquin (Sigma-Aldrich, Darmstadt, Germany) for 30 min at 37 °C (5% CO₂) in isotonic solution containing (in mM) 140 NaCl, 5 KCl, 1.2 CaCl₂, 0.5 MgCl₂, 5 glucose, and 10 Hepes (300 milliosmoles/liter, pH 7.4), plus different concentrations of Zn²⁺ and/or chloroquine (CQ).Cells were then dissociated with Trypsin 0.05% in 0.53 mM EDTA, and were washed with PBS 1x. Fluorescence was quantified using an LSRII flow cytometer. Further analysis was performed using Flowing software (Perttu Terho, Turun yliopisto, Turku, Finland).
For in vivo confocal imaging, cells grown on 22 mm coverslips were incubated with Lysotracker, together with FluoZin-3AM or Zinquin, in a solution with 50 µM Zn²⁺ for 30 min; they were then washed twice with PBS and placed under the microscope in isotonic solution for imaging with an SP8 Leica microscope (Wetzlar, Germany).

Western blotting was performed as described [34 (link)] using antibodies directed against the FLAG epitope (M2 monoclonal antibody, Sigma-Aldrich, catalog number A8592, working dilution 1:5000), ERK2 and phosphoERK1/2 (Santa Cruz Biotechnology, catalog numbers sc-1647 and sc-16982, working dilution 1:7500 and 1:5000, respectively), MITF (Cell Signaling Technology, Danvers, MA, USA, catalog number 12590, working dilution 1:3000), or β-actin (Sigma-Aldrich, catalog number A2066, working dilution 1:10,000). For MC1R immunostaining and signal quantification, 3 × 10⁴ cells were seeded on glass coverslips and grown for 48 h. Confocal MC1R detection was performed as previously described [33 (link)]. Briefly, after 10 min fixation with 4% paraformaldehyde in PBS, cells were permeabilized using a 15 min treatment with 0.4% Triton X-100 in PBS (if needed). MC1R was labeled after overnight incubation with anti-flag rabbit antibody (1:400 in PBS containing 4% BSA) followed by a 1h incubation at room temperature with Alexa 488-conjugated secondary antibody (ThermoFisher, catalog number A-11070, 1:700). For non-permeabilized cells, all steps after fixation were performed over an ice bed. Samples were mounted with DAKO medium (Glostrup, Denmark), and confocal micrographs were obtained with the SP8 Leica microscope. Around 100 randomly selected cells per condition were quantified with ImageJ.