Confocal microscopy, live cell imaging, and intravital microscopy

Confocal microscopy

LmSd-RFP-infected mouse ears were mounted in Tissue-Tek OCT (Sakura Finetek). Cryostat cut sections (7 μm) were placed onto Superfrost Plus microscope slides (Fisher Scientific) and fixed in 4% paraformaldehyde for 10 min. For immunohistochemistry, the sections were blocked with 10% FCS in PBS and incubated with Alexa647 anti-mouse SiglecF (E50-2440, BD Biosciences) and Alexa488 anti-mouse CD206 (C068C2, Biolegend). After 1 h incubation at 37°C in a humidified chamber, the slides were washed in PBS for 10 min and stained with DAPI (ThermoFisher) and mounted with ProLong® Gold Antifade mountant (ThermoFisher). The samples were analyzed with a Leica SP8 confocal laser fluorescence microscope and Imaris software (Bitplane).

Live cell imaging 

BMDMs were plated at 1 x 10⁴ / well in Lab-Tek Chambered #1.0 Borosilicate Coverglass System (Nunc) and stimulated with 10 ng/ml IL4/10 for 72 hours. For live imaging, BMDMs were labeled with CellTrace Far Red kit (ThermoFisher Scientific). DsRed⁺ eosinophils were differentiated from bone marrow progenitors of Actb-DsRed.T3 mice. GFP⁺ PMNs were isolated from bone marrows of Lyz2^GFP mice using Neutrophil isolation kit (Miltenyi Biotec). DsRed⁺ eosinophils and GFP⁺ PMNs were added, each at 1 x 10⁴ / well, to fluorescently labeled BMDMs in the presence of anti-CCL24 Ab or isotype control (R&D systems). For imaging peritoneal cells ex vivo, isolated cells were incubated with an anti-Fc-γ II/III (CD16/32) receptor Ab (2.4G2, BD Biosciences) in PBS containing 1% FCS followed by Alexa594 anti-mouse F4/80 (BM8, Biolegend) and Alexa647 anti-mouse Siglec-F (E50-2440, BD Biosciences) antibodies for 1 hour on ice. Peritoenal isolates were plated at 2.5 x 10⁵ / well in Lab-Tek Chambered #1.0 Borosilicate Coverglass System (Nunc). Live-cell microscopy was performed using Leica DMi8inverted 5 channel confocal microscope equipped with ultra-sensitive hybrid detectors (HyDs, Leica Microsystems) and an Environmental Chamber (NIH Division of Scientific Equipment and Instrumentation Services) to maintain 37° C and 5% CO₂. Argon laser for 488 nm excitation, and HeNe lasers for 594 and 633 nm excitation were used at minimal laser power (0.2-1%). For time-lapse recording, Z stacks of cellular monolayer (Z=10–15 µm) were collected over time (1 to 12 h) in multiple positions using Mark-and-Find application of Leica Application Suite X (LAS X).  Post-acquisition image processing was performed using Imaris software (Imaris version 9.2.1, Bitplane AG, Zurich, Switzerland).

Intravital microscopy

C57BL/6 mice or eoCre il4/13^f/f mice were intravenously injected with 20g of eFluor450 anti-mouse CD31(390, Invitrogen) or 0.5 % Evans Blue dye to outline blood vessels, and with 25g Manocept-Cy5 or -Alexa488 to visualize dermal TRMs, immediately prior to imaging.Non-invasive intravital imaging of the mouse ear was performed using Leica DMi8 inverted confocal microscope (Leica Microsystems) with dual multiphoton lasers Mai Tai and InSight Deep See (Spectra Physics), and full range of visible lasers. Additionally, the microscope was equipped with external and internal HyDs; Lx25.0 water-immersion objective with 0.95 NA (Leica Microsystems) and 2 mm working distance; a motorized stage; and Environmental Chamber (NIH Division of Scientific Equipment and Instrumentation Services) to maintain 37° C. Anesthesia was induced with 2 % Isoflurane (Baxter) and maintained at 1.5 % during imaging. A temperature sensor was positioned on the stage near the animal. Mai Tai was tuned to 880 nm excitation; InSigth was tuned to 1150 nm. Diode laser was used for 405 nm excitation; Argon laser for 488 nm excitation; DPSS laser for 561 nm excitation; and HeNe laser for 633 nm excitation wavelengths. All lasers were tuned to minimal power (between 1 and 5 %). For time-lapse imaging, small tiled images of 2x2 fields were recorded over time, and for static images 5x5 fields were acquired using Tilescan application of LAS X. Z stacks consisting of 6-8 single planes (3-5 μm each over a total tissue depth of 50-70 μm) were acquired every 45 seconds for a totalobservation time between 1 to 6 hoursfor 4D reconstruction, surface modeling and tracking with the Imaris software (Imaris version 9.2.1, Bitplane AG, Zurich, Switzerland). Cells were segmented as 3D surface model and tracked using Imaris autoregressive tracking algorithm. Cell tracks are shown as cylinders. Contacts between cells were calculated using “Kiss and Run Analysis” extension for Imaris. Distance Transformation outside of the target surface object method was used to determine closest surface to surface distance of the tracked objects.

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