However, the precise dose of Poly(I:C) may require few adjustments. The rationale of using i.n instillation of Poly(I:C) is that COVID-19 is a pulmonary disease. brainstem. Serial sections, 4 m thick, were performed using the Leica BOND-MAX system (Leica Biosystems Newcastle Ltd., Newcastle upon Tyne, UK), and selected sections were stained with H&E for light microscopy examination. The Leica Bond Polymer Refine Detection System Kit (DS-9800, Leica Biosystems Newcastle Ltd., UK) was used for detection and hematoxylin for counterstaining. For 3, 3-diaminobenzidine (DAB) and H&E staining, pictures were taken using the Olympus microscope (BX60, Serial No. 7D04032) equipped with the microscopes camera (Olympus DP73, Serial No. OH05504) at objective magnifications of 4, 10 and 20. For immunofluorescent staining, images were acquired using the panoramic MIDI II slide scanner (DHISTEC3) at an objective magnification of 1 1.3. 2.7. SARS-CoV-2 Immunohistochemical Staining Immunolabeling of SARS-CoV-2 was performed as previously described [31]. Briefly, paraffin sections were incubated with rabbit SARS-CoV-2 primary antibody (in-house preparation of rabbit polyclonal anti-RBD) diluted 1:800 (for DAB staining) or 1:200 (for immunofluorescent staining) in antibody cocktail solution. For DAB immunohistochemical staining, a goat anti-rabbit horseradish peroxidase (HRP) polymer was applied for 10?min (RT), followed by incubation for 5?min with DAB. For immunofluorescent staining, slides were incubated with anti-rabbit Alexa Fluor 488 secondary antibody (Molecular Probes, Burlington, ON, Canada) in antibody cocktail solution for 1 BTZ043 (BTZ038, BTZ044) Racemate h at RT. 2.8. Histological Evaluation Lung and brain histopathological severity score analysis was performed using a semiquantitative grading scale of 4 points, as follows: Grade 0the tissue appears normal; Grade 1minor pathological findings; Grade 2mild pathological findings; Grade 3moderate pathological findings; Grade 4severe pathological findings [33]. The intensity of the immunostaining reaction for the SARS-CoV-2 antigen was analyzed using a semiquantitative grading scale of 5 points for the intensity and number of positive stained cells, per a 20 field, as follows: Grade 0no positive reaction; Grade 1only few cells are immune positive ( 5 cells); Grade 2very mild immunoreaction (5C15 positive cells); Grade 3mild immunoreaction (15C25 positive cells); Grade 4moderate immune reaction (25C50 positive cells); Grade 5marked immune reaction ( 50 positive cells). 2.9. Percentage of Air Area Calculation Morphometric analysis of H&E images for calculations of air space ratio was determined using brightness segmentation in MATLAB software version 2020a. A BTZ043 (BTZ038, BTZ044) Racemate representative image of each animal in the affected area at the same magnification (10) was taken. Color threshold parameters were determined and remained consistent throughout the analysis. Total area values were measured separately for air BTZ043 (BTZ038, BTZ044) Racemate space and tissue. Percentage of air area was calculated for each image. A total of 5 animals per group were used. 2.10. Flow Cytometry Lungs were harvested on the indicated days post infection and processed using the Gentle MACS machine (Miltenyi, Bergisch Glabach, North Rhine-Westphalia, Germany) according to the manufacturer protocol. Red blood cells were lysed using RBC lysis buffer (BD Biosciences, Franklin lakes, New Jersey, U.S.), and cell suspensions were washed in PBS and resuspended in flow buffer (PBS supplemented with 2% FBS and 0.05% NaN3). Staining for extracellular markers was performed using the antibodies listed below for 20 min at 4 C before fixation in 3% paraformaldehyde for 30 min at RT. Samples were collected on an LSR-Fortessa flow cytometer Mouse monoclonal to ALCAM (BD BioSciences), and data were analyzed using FlowJo software v10 (Tristar, CA, USA). The following mAb clones were used for staining: CD3 (145-2C11), NK1.1 (PK136), CD11c (N418) and SiglecF (1RNM44N). All antibodies were purchased from Thermo Fisher (San Diego, CA, USA). Aqua Live/Dead cell stain (Thermo Fisher) was used for exclusion of dead cells. 3. Results 3.1. Poly(I:C) Treatment Protects Mice against a Lethal SARS-CoV-2 Infection The therapeutic effect of Poly(I:C) against a lethal dose of SARS-CoV-2 was tested with the transgenic K18-hACE2 BTZ043 (BTZ038, BTZ044) Racemate mouse model [34]. Mice were infected by intranasal (i.n.) instillation with 500 PFU of SARS-CoV-2 and treated i.n. with Poly(I:C) either at the time of infection (0 h post infection (hpi)), 6 or 12 hpi. Body weight changes (Figure 1a) and mortality (Figure 1b) were daily monitored for 17 days. Infected untreated mice started losing weight at 3 dpi and succumbed to infection 3C6 days later. However, mice infected and.