Ec plan neofluar 20x 0.50 objective

Frozen ENL skin lesion sections assays were performed in a Leica LM3000 cryostat, fixed in acetone, and hydrated in 0.01 M PBS. Unspecific binding sites were blocked with 10% Normal Goat Serum (NGS; Sigma-Aldrich) and 5% Fetal Calf Serum (FCS, GIBCO, Life Technologies) in 0.01 M PBS for 1 h at room temperature. Mouse IgG1 anti-human CD64 (1:50; Biolegend, 305002) and rabbit IgG anti-human MPO (1:100; Santa Cruz Biotechnology, SC-16128-R) and their respective isotypes were diluted in 1% Bovine Serum Albumin (BSA, Sigma-Aldrich), 1% NGS in 0.01 M PBS and incubated at 4°C overnight. Tissue sections were washed 3 times and incubated with Alexa Fluor 532 goat anti-mouse IgG (1:1000, ThermoFisher Scientific, A-11002) and Alexa Fluor 633 goat anti-rabbit IgG secondary antibodies (1:1000, ThermoFisher Scientific, A-21070) for 1:30 h at room temperature. The nuclei were stained with 4′-6-diamidino-2-phenylindole (DAPI; 1:10000, Molecular Probes, D1306), and slides were mounted with VECTASHIELD Mounting Medium (Vector Laboratories, H-1000). Tissues were imaged using an Axio Observer.Z1 fluorescence microscope equipped with a Colibri.2 illumination system (Carl Zeiss, Oberkochen, Germany) and the EC Plan-Neofluar 20x/0.50 objective and Plan-Apochromat 63x/1.3 oil objective. Images were acquired with a digital camera AxioCam HRm and AxioVision Rel. 4.6 software (Carl Zeiss).

EpiAirway™ tissues were obtained from MatTek. Upon delivery, the tissues were processed according to the supplier's protocol. For infection, PBS washed bacteria obtained from RPMI-CAS mid-log cultures were applied to the apical surface (MOI 100) in presence of either ASN-1, ASN-2, the mixture of these mAbs or an isotype control mAb (2 µM each) in 30 µL volumes. MAbs were also added to the basal feeding medium at the same concentration. Following incubation for 24 hours at 37 °C and 5% CO₂, the basolateral medium was harvested for measuring LDH release using the CytoTox-ONE™ Homogeneous Membrane Integrity LDH release Assay (Promega) and for subsequent measurement of PMN toxicity in a Cell Titer-Glo® Luminescent Cell Viability Assay Kit (Promega) as described above. Saponin in dH₂O (1%) was used as lysis control in LDH release assays. Bacteria on the apical surface of the tissue were collected by three washing steps with 300 µL PBS and serial dilutions were plated on sheep blood agar plates for CFU determination. The washed tissues were fixed overnight with a total of 2 mL 4% paraformaldehyde (Sigma) at 4 °C, embedded in paraffin and 1 µm sections were stained with hematoxylin and eosin at the automated slide stainer Gemini AS (Histocom). Images were taken with a ZEISS EC Plan-Neofluar 20x/0.50 objective on a ZEISS Imager M1 microscope equipped with AxioCamMRc5 color camera.